ORIGINAL ARTICLE

Timothy Lovell Æ Jian Li Æ David A. Case

Louis Noodleman

FeMo cofactor of nitrogenase: energetics and local interactionsin the protein environment

Received: 12 September 2001 /Accepted: 17 January 2001 / Published online: 23 March 2002� SBIC 2002

Abstract A combined broken-symmetry density func-tional and continuum electrostatics approach has beenapplied to the iron-molybdenum center (FeMoco) ofnitrogenase to evaluate the energetic effects of the localamino acid environment for several spin alignments ofFeMoco. The protein environment preferentially stabi-lizes certain spin coupling patterns. The lowest energyspin alignment pattern in the protein displays calculatedproperties that match the experimental data better thanany of the alternative possibilities. The total interactionenergy of the protein with FeMoco has been evaluatedand the contribution of each amino acid residue hasbeen broken down into sidechain and backbone com-ponents. Arginine, lysine, aspartate and glutamatesidechains exert the largest electrostatic influence onFeMoco; specific residues are highlighted and their in-teraction with FeMoco discussed in the context of theavailable X-ray data from Azotobacter vinelandii (Av).Observed data for the MN(resting state)fiMOX(one-electron oxidized state) and MNfiMR(one-electronreduced state) or MI(alternative one-electron reducedstate) redox couples are compared with those calculatedfor Av. The calculated redox potentials are fairly in-sensitive to the spin state of the oxidized or reducedstates and the predicted qualitative trend of a morenegative redox potential for the more reduced MNfiMR

or MI couple is in accord with the available redox data.These calculations represent a first step towards thedevelopment of a microscopic model of electron andproton transfer events at the nitrogenase active site.

Keywords FeMo cofactor Æ Nitrogenase ÆDensity functional calculations Æ Continuumelectrostatics Æ Azotobacter vinelandii

Introduction

Molybdenum-iron nitrogenase [1, 2, 3] consists of twoseparately purified proteins, the Fe protein and theMoFe protein, named according to their constituentmetal ions [4]. The Fe protein contains a single 4Fe4Scluster similar in composition to that observed in otheriron-sulfur electron transfer proteins. The MoFe proteincontains two unique polynuclear metal-sulfur clusters,the P cluster (stoichiometry 8Fe7S) and the catalytic Mcenter or FeMo cofactor (stoichiometry Mo7Fe9S.R-homocitrate). The molybdenum-dependent nitrogenasehas been the focus of extensive research for many years[5, 6] and the reduction of the primary substrate (N2) isbelieved to take place at the FeMo cofactor in the a-subunit of the MoFe protein [7, 8]. In addition to thismore well-known, physiologically relevant conversion ofN2 to NH3, the ATP-dependent reduction of other smallmolecules (C2H2, HCN and CO) having multiple bondsalso occurs. However, the major challenge posed by thisenzyme from the chemical point of view still remains:how are N2, 8H

+ and 8e– combined by FeMoco to give2NH3 and H2 under ambient conditions and at biolog-ical redox potentials?

Despite the solution of the X-ray structure [9, 10, 11,12, 13, 14, 15, 16, 17] of the MoFe protein revealing theunique structure of FeMoco in its resting form, ratherlimited knowledge of cofactor function at the atomiclevel still prevails. There are presently no experimental X-ray structures for the more reduced turnover states and itappears that the protein environment provides FeMocowith ample room to adopt a wide variety of possiblestructures. It is therefore not surprising that agreementconcerning the mechanism of dinitrogen activation andthe evolution of dihydrogen (both general and obliga-tory) has not yet been reached. Significant insight into

J Biol Inorg Chem (2002) 7: 735–749DOI 10.1007/s00775-002-0348-0

T. Lovell (&) Æ J. Li Æ D.A. Case Æ L. Noodleman (&)Department of Molecular Biology TPC-15,The Scripps Research Institute,La Jolla, CA 92037, USAE-mail: tlovell@scripps.eduFax: +1-858-7848896E-mail: lou@scripps.edu

Present address: J. LiJohnson & Johnson Pharmaceutical Research & Development,PO Box 776, Welsh and McKean Roads,Spring House, PA 19477-0776

this problem may be gained by carrying out ab initiodensity functional calculations [18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33] on a quantummodel thatfaithfully represents the active site of the protein. Thecaveat is, however, that the active site must not be solarge that the calculations become intractable at the highlevel of theory required to afford reliable energetics.

In a previous article [34], a working model of Fe-Moco and its immediate protein environment was con-structed (Fig. 1). In the resting state MN, the clustermodel comprised the [Mo4+6Fe2+Fe3+9S2–]+ core, thesidechain ligands of Cys275 represented by a methyl-thiolate (charge=–1), His442 represented by an imi-dazole (charge=0) and the fully deprotonatedhomocitrate ligand (charge=–4) within the firstcoordination shell, giving an overall total cluster ofcharge –4. A number of second shell ligands have alsobeen included in the model: the sidechain of Gln191(represented by acetamide) and four structurally char-acterized water molecules (Wat639, Wat610, Wat631and Wat633 from 2MIN), which are the hydrogenbonding partners to the negatively charged oxygen endsof the homocitrate ligand. Broken-symmetry (BS) den-sity functional theory (DFT) calculations were

performed to examine the best characterized states of theFeMo cofactor [7, 8]. These states included the one-electron oxidized state, MOX [35, 36, 37, 38, 39], theresting state, MN [38, 39], the catalytically observedone-electron reduced state, MR [40], and a radiolyticallyreduced state, MI [40].

For the resting state MN, several spin alignmentswere examined1 (denoted BS2, BS6, BS7, etc., in Fig. 2)that satisfied the S=3/2 total spin requirement of thecluster. In the gas phase, BS2 and BS6 were found to bepreferable by energetic criteria, lying within 4 kcal/molof each other. With its 2:1 pattern of site spins within theMo3Fe triangle, state BS6 gives rise to three possiblespin isomers, each being obtained by cyclic permutation(�120� rotation about the three-fold axis) of the sitespins within the Fe3Fe4Fe5 triangle. The relative ener-getic ordering of these low-lying states in the gas phasewas found to be BS2<BS6-3<BS6-1<BS6-2. The dis-tinct preference of FeMoco to favor these four spinalignments was traced to the number of antiferromag-netic (AF) interactions between site Fe2¢ and sites Fe6¢,Fe7¢ and Fe8¢. BS7 and all other spin alignment patternsdisplayed fewer AF interactions involving Fe2¢ and werefound to be even higher in energy relative to BS6-2.

Having examined the properties of a number of low-lying states of the isolated FeMoco in detail, ourattention now turns towards understanding how thestructural features of FeMoco relate to the observedcatalytic function in the presence of the protein andsolvent environment. In this contribution, the quantumactive site model depicted in Fig. 1 is embedded in anelectrostatic/dielectric representation of the protein andsolvent environment [41]. This allows the energetic ef-fects of the protein and solvent environment on each BSspin state and other properties to be assessed, andhighlights the important role that the protein and sol-vent environment plays in regulating the energetics at

Fig. 1. The model of the FeMoco active site cluster and itssurrounding protein environment. Atoms are identified by color:Fe: magenta; S: yellow; Mo: green; O: red; N: blue; C: dark gray;H: white. The figure was prepared using MOLSCRIPT [66]

1To describe spin polarization and spin coupling, the calculationswere done with the spin-unrestricted BS approach. The BS state isnot a pure spin state, but rather a mixed state in which the majorityspin and minority spin are arranged either spin up or spin down togive a spin coupling pattern with the correct net total spin andeither an overall antiferromagnetic or ferromagnetic alignment. Toconstruct a desired BS state for the resting FeMoco, a calculationon the high-spin (HS) state was first completed, which is a pure spinstate described by a single determinant, with all unpaired electronsaligned in the same direction (spin up) to adopt the highest possibletotal spin state S. For the Mo4+6Fe2+Fe3+ oxidation state, the HSstate had total spin S=29/2. The density of the HS state was thenmanipulated by exchanging designated blocks of a and b electrondensities. In this way, the starting density for the desired spin-flipped S=3/2 state was created, from which BS states were ob-tained by SCF convergence. Similarly, calculations have also beenundertaken for the spectroscopically characterized diamagneticstate MOX, which lies one-electron oxidized relative to MN, as wellas the catalytically and radiolytically observed one-electron re-duced species, MR and MI, respectively. For MOX, spin states ofS=0, 1 and 2 were constructed, with S=0 generated from S=3/2by removal of one a electron followed by a single spin transition togive a state with an equal number of a and b electrons. MOX (S=1)has an excess of two a spin electrons and MOX (S=2) has an excessof four a spin electrons. Similarly, MR (S=2) and MI (S=1) haveexcesses of four and two a spin electrons, respectively, and no spintransitions are required

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the active site. In addition, the breakdown of the inter-action energy between particular amino acid residuesand the two most favorable BS states for FeMoco withinthe Azotobacter vinelandii protein environment thatfollows provides a probe of the structure-function rela-tionship relevant to site-directed mutagenesis studies.Finally, in accord with our previous methodology usedto calculate redox potentials of 2Fe2S clusters in pro-teins [42], observed redox data [43, 44, 45] are comparedwith calculated redox data in the protein environmentfor the characterized states (MOX, MN, MR and MI) ofthe FeMo cofactor.

Methods

Protein structures and optimization of the H-bonding network

X-ray crystallographic coordinates have been reported for nitro-genase from several bacteria. They now include Azotobacter

vinelandii (Av) [9, 10, 11], originally refined to 2.2 A (PDB code:1MIN), subsequently to 2.0 A [PDB codes: 2MIN (ox), 3MIN(red)] [17]; Clostridium pasteurianum (Cp1) [13] to 3.0 A (PDB code:1MIO); and most recently, Klebsiella pneumoniae (Kp) [15] to 1.6 A[PDB codes: 1QH1 (ox), 1QH8 (mixed), 1QGU (red)]. The MoFeproteins of Kp and Cp are structurally and functionally homolo-gous to Av. The results from calculations using the Kp structuresare on the whole very similar and for historical reasons, and con-sistency with our previous paper [34], the 2MIN and 3MIN pro-teins are reported in this work.

The MoFe protein of Av is an a2b2 tetramer,Mr�250,000, the aand b subunits of which are composed of 491 and 522 residues,respectively. The ab dimer subunits are related to each other by anon-crystallographic axis of twofold symmetry. An examination ofthe 2MIN and 3MIN X-ray structures reveals that close contactsbetween pairs of ab subunits are exclusively between the b subunits,an important feature as communication between FeMoco clustersand long-range binuclear Mo chemistry is thus prevented. The a2b2tetramer contains 30 Fe atoms and two Mo atoms, distributedbetween two P clusters {which are paramagnetic with total clusterspin S=3 or 4 (2MIN) [46] in the oxidized (POX) state and S=0(3MIN) in the reduced (PN) state} and two FeMoco centers (whichare paramagnetic and give rise to an S=3/2 EPR signal) [38]. Eachab subunit functions independently of the other and therefore

Fig. 2. Spin coupling align-ment for the 10 simple broken-symmetry states of the FeMococluster at the Mo4+6Fe2+Fe3+

oxidation level. Relative ener-gies of the active site quantumcluster in vacuum are given

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contains one P cluster and one FeMoco. There is minimal sequencehomology between the a and b subunits, although similar poly-peptide folds are noted to comprise three domains of primarily ahelix and b sheet type. At the interface between the three domainsin the a subunit is a wide and shallow cleft and the FeMoco oc-cupies the bottom of this cleft. The P cluster is found at the abinterface with a pseudo-twofold rotation axis relating the two absubunits [4]. The active monomeric domain used in our calculationswas chain A of Av shown in Fig. 3. The b chain and the P clusterare omitted for clarity. For our calculations, the a chain of 2MINcomprises 480 amino acid residues.

2MIN and 3MIN were crystallized under experimental condi-tions of around pH 7.0 [9, 10, 11]; to simulate these conditions inan approximate way for each protein, hydrogen atoms in theprotein were added at pH 7.0. The His442 sidechain coordinatedthrough Nd1 to Mo was protonated at N�2. The positions of thehydrogens were then optimized using standard InsightII (Discovermodule, BioSym/MSI) [47] minimization procedures, using formalmetal charges (3+/2+) assigned to the Fe sites (prior to quantumDFT calculations), Mo4+ for molybdenum and S2– for sulfides,while the total charge for each single amino acid residue was as-signed the appropriate integer charge (+1, 0 or –1) as a sum of thepartial atom charges obtained from the standard InsightII aminoacid fragment libraries. During the hydrogen atom optimization,all heavy atom positions were kept fixed.

As indicated in our previous paper [34], starting geometries forthe active site quantum models were taken from the 1MIN struc-ture. As the hydrogen bonding interactions of FeMoco and theimmediate protein environment are important, the dipoles of thewater molecules were oriented favorably to interact with the dan-gling oxygen ends of the homocitrate and with water moleculesthemselves. Final geometries were computed by density functionalmethods. The active site FeMoco model is considered as a smallpart of the protein in which the appropriate linking C atoms of theHis442 (Cb), Cys275 (Ca) and Gln191 (Cb) amino acid sidechainswere replaced by H atoms to saturate open valencies. These junc-tion H atoms lie at the interface of the quantum and classical

regions and were treated in an appropriate manner to maintaincharge conservation. A brief description of the method used toassign the molecular electrostatic potential charges is given below;the theoretical foundations of the approach are documented else-where [42, 48, 49].

Electrostatic potential charges

Molecular electrostatic potential (ESP) charges were generated byusing a modified version of the CHELPG code of Breneman andWiberg [48]. This set of point charges represents a best fit of thequantum electrostatic potential [49]. The net charge of the activesite cluster and the three cartesian dipole moment componentsfrom ADF [50] calculations were used as constraints. The fittedpoints lay on a cubic grid within the envelope between the van derWaals radius and the outer atomic radius, with a grid spacing of0.2 A. The outer atomic radius for all atoms used was 5.0 A andthe van der Waals radii for Mo4+, Fe2+/3+, S, N, C, O and H were1.70, 1.50, 1.80, 1.55, 1.67, 1.40 and 1.20 A, respectively. In orderto minimize the uncertainties in the fitting procedure and thus re-duce the sensitivity of the charges to noise in the target electrostaticpotential, the singular value decomposition (SVD) method [51] wasused to obtain a model with stable atomic charges and an accuratemolecular dipole moment. Junction H atoms were excluded fromthe charge fitting procedure by setting these charges to zero, butthey were included in the construction of the fitting grid envelope.The corresponding Ca and Cb junction atoms were also included todetermine the low dielectric (�=1) region. For all other atoms inthe protein the PARSE charges [52] and radii were assigned.

Modeling the protein/solvent environment

The active site cluster with optimized geometry [34] was dockedback into the protein using the Xfit program [53], using a least-squares fitting scheme for the atoms of the active site cluster. Thenthe MEAD program [54, 55, 56, 57] (version 1.1.8) was employedto calculate the electrostatic energies in a multi-dielectric model.Version 1.1.8 is available through the internet via anonymous ftp toftp.scripps.edu under the directory ‘‘electrostatics’’ or through theBashford web page at http://www.scripps.edu/bashford. A detailedanalysis of the resulting reaction field and protein field energy termscan be found elsewhere [42]. The dielectric constants are 1, 4 and 80for active site, protein and solvent regions, respectively. The di-electric constant used for the protein region accounts for somereorientational relaxation of the protein in an approximate way [58,59, 60]. For the purpose of defining the dielectric boundary, atomicradii for each of the atoms defined previously were chosen. TheseN, C, O and H radii are very similar to the PARSE radii suggestedby Sitkoff et al. [52] and are widely used for protein electrostatics.The ‘‘solute interior’’ was defined as the region inaccessible to anypart of a probe sphere of radius 1.4 A rolling on the molecularsurface of the atomic spheres. The resulting Poisson equation wassolved by using an over-relaxation algorithm on successively finergrids of size 613, 813 and 1013 with linear spacings of 1.0, 0.25 and0.15 A, respectively. In the present paper the one-step approach,rather than the iterative self-consistent reaction field (SCRF)methodology, was used to calculate protein field and reaction fieldenergies [42, 48, 49]. For comparison with the protein results, thesolvation energies of the active site clusters in pure solvent (di-electric constants of �=80 and 190 to simulate water and NMF,respectively) have also been evaluated.

Results

ESP charges

In Table 1 we present ESP charges for states BS2, BS6-1,BS6-2, BS6-3 and BS7 of FeMoco. In the context of the

Fig. 3. The MoFe cluster in the a chain of the 2.0 A structure ofthe MoFe protein of Azotobacter vinelandii nitrogenase. The Pcluster and b chain are omitted for clarity. The figure was preparedusing MOLSCRIPT [66]

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modeling of the nitrogenase protein, these charge setsmayprove a useful starting point for further modeling of theresponse of the protein environment to the charge distri-bution of FeMoco calculated from quantum mechanics.

For the resting MN state, on a per atom basis the ESPcharges are calculated to be much less than the formalcharges. This difference is most evident for the transitionmetal sites, i.e., Fe2+, Fe3+ and Mo4+. ESP charges ofsimilar magnitude (ranging from 0.14 to 0.32) are calcu-lated for sites Fe3, Fe4, Fe5, Fe6¢, Fe7¢ and Fe8¢, reflectingthe similarities in the ligand environment and associatedspin density. The ESP charge associated with site Fe2¢(�0.60) is higher than any of the other Fe sites across allfive BS states, consistent with Fe2¢ being themost oxidizedsite and fully in accord with the energy level ordering andspin density analysis noted previously for BS6 [34].

The ESP charges within FeMoco are also in contrastto their idealized equivalent in which pure ionic bondingprevails. In such a situation, the total electron count isderived from a consideration of the formal charges:Fe3+/Fe2+ for a ferric/ferrous ion, S2–/SR– for inor-ganic sulfide ligands and the methanethiolate Cys275,respectively, RCO2

– for the terminal carboxylates of thehomocitrate and 0 for the imidazole sidechain of His442.The difference between the ideal and the actual ESPcharges provides an indication of covalency effectswithin the active site, particularly when compared to4Fe4S clusters such as Fe4S4(SMe)4

2– (S=0), one-elec-tron reduced ferredoxin (S=7/2) and the two-electronreduced ferredoxin (all-ferrous cluster, S=4) (TorresRA, Lovell T, Noodleman L, Case DA, manuscript inpreparation; Liu T, Noodleman L, Case DA, unpub-lished results) (S=7/2 was chosen as the reduced 4Feferredoxin rather than the usually observed S=1/2 formbecause S=7/2 resembles the 4Fe¢ cubane part of theFeMoco BS6 states). Regardless of which 4Fe4S cluster

the comparison is made to, the ESP charges associatedwith the Fe and lS sites in FeMoco are generally muchlower in absolute value (for BS6-1, ESP Feav=0.30; ESPlSav=–0.46) than those calculated for the simple cu-banes [in reduced ferredoxin (S=7/2), ESP Feav=0.68;ESP lSav=–0.68]. The Fe-S bonds are therefore con-siderably less polar in FeMoco, suggesting enhancedmetal-ligand interactions and a degree of charge cova-lency not observed in the simple cubane counterparts.

A qualitative picture of the accepting orbitals for MN

has been described in detail elsewhere for state BS6-1[34]. Briefly, BS6-1 displays spin-up and spin-downLUMOs that contain atomic contributions primarilyfrom Mo4+ and the homocitrate, and from the centralprismane Fe sites and lS2 atoms, respectively (spin upand spin down are aligned, and oppositely aligned, tothe net spin of S=3/2 of MN, respectively). Thesequalitative features are consistent with observationsfrom Mossbauer spectra of the one-electron reducedstates [40]. The ESP charges associated with FeMoco inresponse to the one-electron redox process are also givenin Table 1 for states MOX (S=2, 1 and 0), MR (S=2)and MI (S=1). Using state BS6-1 as the appropriatereference state, when the active site undergoes a singleelectron transfer event, the principal question that wehave tried to address is: where does the extra chargebecome redistributed around FeMoco? The change inthe ESP charges associated with the six trigonal Fe siteson oxidation or reduction amounts to approximately25% of the total charge change on redox. Such smallchanges in charge suggest that all six Fe sites act inconcert to share the charge change on accepting or los-ing an electron. Charge is redistributed around FeMocosuch that no one trigonal Fe site stands out as being thespecific site for oxidation or reduction at this redox level.The remaining 75% of the change in the ESP charges on

Table 1. Calculated ESPcharges for low-lying states of

the Mo4+6Fe2+Fe3+ (MN)FeMoco cluster. Also shownare charge sets for MOX, MR

and MI

State Mo1 Fe2¢ Fe3 Fe4 Fe5 Fe6

¢ Fe7¢ Fe8

¢

MOX (S=2) 1.06 0.52 0.12 0.19 0.17 0.21 0.27 0.27MOX (S=1) 1.06 0.51 0.10 0.19 0.19 0.21 0.28 0.26MOX (S=0) 1.08 0.48 0.12 0.21 0.19 0.18 0.25 0.24MN (BS6-1) 1.08 0.61 0.14 0.24 0.23 0.25 0.31 0.29MN (BS6-2) 1.11 0.62 0.17 0.23 0.28 0.24 0.31 0.28MN (BS6-3) 1.10 0.61 0.18 0.21 0.25 0.25 0.31 0.27MN (BS2) 1.12 0.61 0.22 0.28 0.27 0.25 0.32 0.28MN (BS7) 1.09 0.59 0.20 0.24 0.25 0.22 0.30 0.31MR (S=2) 1.17 0.71 0.19 0.26 0.25 0.26 0.35 0.32MI (S=1) 1.16 0.70 0.19 0.23 0.26 0.28 0.36 0.32

S9 SS10 SS11 SS12 SS13 SS14 SS15 SS16 SS17 SS28

MOX (S=2) –0.37 –0.33 –0.40 –0.44 –0.41 –0.34 –0.31 –0.28 –0.32 –0.53MOX (S=1) –0.37 –0.30 –0.38 –0.40 –0.41 –0.37 –0.32 –0.29 –0.31 –0.53MOX (S=0) –0.33 –0.26 –0.33 –0.40 –0.42 –0.38 –0.34 –0.29 –0.33 –0.55MN (BS6-1) –0.48 –0.41 –0.49 –0.53 –0.52 –0.47 –0.39 –0.34 –0.38 –0.62MN (BS6-2) –0.46 –0.41 –0.51 –0.53 –0.53 –0.47 –0.41 –0.39 –0.37 –0.61MN (BS6-3) –0.47 –0.42 –0.48 –0.53 –0.51 –0.48 –0.39 –0.38 –0.39 –0.62MN (BS2) –0.48 –0.42 –0.47 –0.53 –0.56 –0.45 –0.43 –0.42 –0.43 –0.61MN (BS7) –0.44 –0.39 –0.49 –0.54 –0.53 –0.47 –0.40 –0.37 –0.38 –0.63MR (S=2) –0.55 –0.51 –0.58 –0.66 –0.64 –0.55 –0.46 –0.41 –0.47 –0.70MI (S=1) –0.55 –0.53 –0.58 –0.64 –0.65 –0.57 –0.47 –0.40 –0.45 –0.71

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conversion to MOX and MR or MI occurs at the lS2 andlS3 sites. It is therefore likely that as the Fe sites ofFeMoco become more reduced, the S atoms attain morenegative charge, increasing the likelihood of the S sitesfor subsequent protonation; as the Fe sites of the clusterbecome more oxidized, the S sites become more positiveand the cluster is more likely to release protons. Theseresults indicate that electron relaxation effects are large.Although the active LUMO orbitals have considerableFe and Mo character, the positive charge on these cen-ters increases on reduction of MN, while an increasedelectron density resides mainly on the bridging and ter-minal sulfurs.

Interaction with protein environment

DFT calculations including the first-shell ligands in ourquantum model reproduce the main features observedfor FeMoco. As a next step towards a more completepicture, the electrostatic and polarization interactionstaking place within the long-range protein and solventenvironment are identified for each of the 10 simple BSstates and the additional two states arising from spinisomerization within state BS6. The calculations onlyincorporate the a chain of the MoFe protein, as thisappears the most influential component of the energet-ics. The amino acid residues from the b chain and thecontribution of the P cluster lying at the ab interface areonly minor and are not included2. For comparison, wehave also performed calculations at the MN level inwhich FeMoco is embedded in pure solvent (H2O andNMF) environments. In all cases, we find the pre-formed cluster is stabilized more favorably in the proteinthan in a pure solvent environment by �20 kcal/mol3.For now, we restrict the discussion to the results of thecalculations in the protein environment.

Table 2 gives the decomposition of the long-rangeprotein and solvent environment (Epr) into protein (Ep)and reaction (Er) field contributions for the 12 BS statesof FeMoco. For each BS state:

Epr ¼ Er þ Ep ¼1

2

XqiU�

reacðiÞ þX

qiUprotðiÞ ð1Þ

In this approach, the cluster is represented by a pointcharge model (an electrostatic potential charge). Here,Er and F*reac(i) represent the energy term and corre-sponding potential, respectively, due to the polarizationof the protein and solvent dielectric induced by thecharge distribution qi on all atoms (i) in the active sitecluster in the protein environment. Large values of Er

are indicative of a fairly strong electrostatic interactionbetween the active site cluster and the protein and sol-vent dielectric polarization. Similarly, Ep and Fprot(i)represent the energy contribution and potential arisingfrom the charge distribution in the protein residuesacting on the FeMoco cluster. The Ep component iscomparatively small and only contributes approximately10% of the total Epr in the Av protein. The majorcomponent for stabilization of FeMoco in the proteinenvironment is the larger reaction field term. However,the magnitude of Ep (ranging from 50 to 60 kcal/mol)suggests that the local amino acid environment plays asubstantial role in the energetics and the stabilization ofFeMoco.Ep varies in magnitude (�10 kcal/mol), depending on

the spin alignment of a particular BS state when dockedin the protein. Ep is largest and negative (stabilizing) forstate BS6-3, but within the set of spin isomers of BS6there is only 5 kcal/mol variation, depending on thespecific spin alignment. The reaction field displays aslightly smaller variation, ranging 6 kcal/mol across all12 BS states. The total variation in Epr is small, within arange of about 6 kcal/mol, with only 4 kcal/mol sepa-rating the 12 possible spin patterns associated with statesBS3 through to BS10.

When the protein energies are combined with the gasphase energies, the variability between states becomesmore pronounced. In Table 3 the total energies (Et) ofthe 12 BS spin alignments of FeMoco in the 2MINprotein environment are given relative to state BS6-3.The total energy is plotted in Fig. 4 as a function of thecumulative effect of variation in energy versus BS spinstate alignment for the five lowest lying states. AlthoughBS2 was calculated as the most stable state in the gasphase, when the effects of the protein and solvent envi-ronment are taken into account, then state BS2 becomesdestabilized relative to state BS6-3. The protein fieldcomponent is primarily responsible for the destabiliza-tion of BS2. The protein and solvent environmenttherefore selects one of the BS6 states over state BS2.

Table 2. Calculated interaction energies (kcal/mol) of the 12 broken-symmetry spin alignments of the Mo4+6Fe2+Fe3+ FeMoco clusterin the 2.0 A A. vinelandii protein environment. The total (protein field+reaction field) (Epr) equals the sum of the protein field (Ep) andreaction field (Er). See text for a detailed discussion of terms

E BS1 BS2 BS3 BS4 BS5 BS6-1 BS6-2 BS6-3 BS7 BS8 BS9 BS10

Ep –50.81 –53.25 –53.05 –55.71 –53.36 –55.09 –54.21 –58.66 –53.34 –54.48 –54.78 –54.56Er –429.11 –425.19 –430.21 –431.05 –429.91 –427.46 –428.24 –425.75 –429.45 –428.89 –431.09 –429.41Epr –479.92 –478.44 –483.26 –486.76 –483.27 –482.55 –482.45 –484.41 –482.79 –483.37 –485.87 –483.97

2Incorporation of the P cluster and the amino acid residues of the bchain does not affect the resultant energetics associated with eachBS state in the protein and the values of the calculated redox po-tentials3For all the spin alignments examined for FeMoco states MOX,MN, MR and MI, the protein environment stabilizes the active sitebetter than a pure solvent environment. The magnitude of thisdifference in solvation capability (protein versus solvent) ranges

from �10 kcal/mol for MOX, MR and MI to 20 kcal/mol for MN

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The relative position of state BS7 is raised slightly by theaddition of the protein and solvent environment andlies to higher energy than any of the BS6 states in eithercase.

Decomposition to individual residues

The important interaction energies profiled in our elec-trostatics breakdown of the 2MIN protein residuesacting on FeMoco state BS6-3 are shown in Fig. 5. Allenergies are in kcal/mol and are relative to a pure pro-tein/solvent dielectric in which Ep=0 (all protein chargesare set to zero). The total electrostatic energy of inter-action of a specific residue with the quantum cluster canbe further broken down into contributions from theamino acid sidechain and backbone and this decompo-sition is shown in Table 4. With respect to the overallnegative charge of –4 on the active site, negative inter-action energies from positively charged residues arestabilizing; negatively charged residues are destabilizing.

The most prominent stabilizing contributions to theprotein field energy come from residues that derive fromarginine and lysine residues: Arg359, Arg96, Lys426,Arg277, Arg361, Arg60 and Arg97. These protein resi-dues represent a major component of the stabilization ofthe active site cluster in the protein and provide some –67 kcal/mol of stabilization in the 2MIN protein via acombination of short-range hydrogen bonding and long-range electrostatics interactions with FeMoco. The totalstabilization associated with the selected 14 residues inTable 4 totals –89 kcal/mol. Thus, the residues high-lighted provide a major contribution to the total proteinfield energy (–59 kcal/mol) provided by all 480 residuesin the protein.

Of the residues outlined in Table 4, the peptide side-chains that have the largest electrostatic effect onFeMocoreside within the local FeMoco environment. Here, allatoms appearing within 4 A of FeMoco are defined as thelocal environment. Protein backbone contributionswithin this 4 A cutoff appear less important. Within thelocal sphere of selected residues in Table 4, the positiveand negative charged sidechain interaction energies arelarger than their backbone counterparts. The sidechaininteraction energies appear for pairs of closely foundresidues (for example, Arg-Asp or Arg-Glu combina-tions) that are oppositely charged but approximatelyequal in magnitude such that their combined electrostaticeffect is one of cancellation. By contrast, protein back-bone interactions, although smaller, make a more cumu-lative contribution to the final protein field energy. Thispattern of pairs of oppositely charged sidechain energiescancelling and backbone energies further adding to theoverall stabilization in the protein appears globallythroughout all 480 residues in the active domain. Back-

Table 3. Relative total energies (kcal/mol) of the 12 broken-symmetry spin alignments of the Mo4+6Fe2+Fe3+ FeMoco cluster in the2.0 A A. vinelandii protein environment. The total energy (Et) equals the sum of the gas phase (Eg) and protein energies (Epr)

E BS1 BS2 BS3 BS4 BS5 BS6-1 BS6-2 BS6-3 BS7 BS8 BS9 BS10

Ep +7.85 +3.57 +5.61 +2.95 +5.30 +3.57 +4.45 0.00 +5.32 +4.18 +3.88 +4.07Er –3.36 +0.56 –4.46 –5.30 –4.16 –1.71 –2.49 0.00 –3.70 –3.14 –5.34 –3.66Epr +4.49 +4.13 +1.15 –2.35 +1.14 +1.86 +1.96 0.00 +1.62 +1.04 –1.46 +0.41Eg +18.85 –3.74 +15.51 +9.26 +20.52 –0.16 +0.38 0.00 +2.29 +6.66 +8.21 +4.99Et +23.34 +0.39 +16.66 +6.91 +21.66 +1.70 +2.34 0.00 +3.91 +7.70 +6.75 +5.40

Fig. 4. Energies of the five lowest-lying broken-symmetry states ofMo4+6Fe2+Fe3+ FeMoco in Azotobacter vinelandii. All energiesare taken relative to state BS6-3 in kcal/mol. All other states liehigher in energy than BS7

Fig. 5. Residue by residue breakdown of the total protein fieldinteraction energy in the 2.0 A Azotobacter vinelandii environment

741

bone contributions are therefore the primary source ofstabilization of FeMoco in the active domain. The localenvironment around FeMoco is of considerable interest.The magnitude of the interaction energies suggests theamino acid sidechains play an important role and areworth a closer examination.

Of these amino acid residues that have favorable in-teractions (Fig. 5), the largest contribution comes fromArg359, which serves to stabilize the cluster via three

important hydrogen bonds to the first-shell inorganicsulfur-based ligands, Arg359.N-H� � �lS2.12 (2.81 A)4,Arg359.Ng2-Hg12� � �lS3.17.His246 (3.35 A) and Arg359.Ng1-Hg21� � �lS2.13.His246 (3.99 A). In Fig. 6, thesehydrogen bonding interactions with FeMoco are shownin more detail and are designated as sidechain (s) orbackbone (b) type interactions. Each hydrogen bondconstitutes an arginine to inorganic sulfur or sulfidecharged-charged interaction, the summed effect of whichprovides –18 kcal/mol of stabilization associated withArg359.Compared toArg359,Arg96 is another positivelycharged residue that provides a substantial degree ofstabilization (–14 kcal/mol) on FeMoco, with the very

strong Arg96.Ng1-Hg12...lS2.13 (2.55 A) and Arg96.Ng1-Hg11...lS2.13 (3.05 A) interactions as its major hy-drogen bonding components. The relative electrostaticcontributions of the Arg96 and Arg359 sidechains are theopposite of what one expects based on purely electrostaticarguments. With its shorter hydrogen bonds to FeMoco,the sidechain of Arg96 would be anticipated to exert the

Fig. 6. Important hydrogenbonding interactions (dashedlines) from the local proteinenvironment with FeMoco.With the exception of His195and Wat617, only the promi-nent stabilizing amino acidresidues identified from ourelectrostatics breakdown(Fig. 5) are indicated. s=side-chain-based interaction withFeMoco; b=backbone-basedinteraction

Table 4. Residue-by-residue b-reakdown of the protein fieldinteraction energy (kcal/mol)

for the Mo4+6Fe2+Fe3+

FeMoco state BS6-3 dockedinto the 2.0 A A. vinelandiiprotein

Residue Total Back Side Residue Total Back Side

Arg359 –18.86 –2.73 –16.13 Asp228 9.51 –0.57 10.08Arg96 –14.06 –0.73 –13.33 Glu380 8.69 –1.34 10.03Lys426 –10.11 –3.63 –6.48 Asp234 8.63 –0.44 9.07Arg277 –8.84 –2.57 –6.27 Asp386 6.93 –0.05 6.98Arg361 –6.77 –1.13 –5.64 Glu427 6.50 –0.94 7.44Arg60 –4.72 –0.94 –3.78 Glu440 5.65 0.09 5.56Arg97 –4.55 –0.86 –3.69 Asp402 3.00 –0.14 3.14Lys68 –3.57 0.00 –3.57 Cys62 2.44 1.37 1.07Gly356 –3.34 –3.34 0.00 Asp403 2.05 –0.61 2.66Gly61 –3.33 –3.33 0.00Gly357 –3.16 –3.16 0.00Ile425 –3.08 –3.08 0.00Leu358 –2.76 –2.76 0.00Cys275 –2.25 –2.25 0.00Total ofresidues

–89.40a –30.51a –58.89a 53.41b –2.62b 56.03b

Total inproteinc

–58.66 –58.96 0.30

aFourteen residues listed above with negative interaction energies are cations or neutralbNine residues listed above with positive interaction energies are anions or neutralcChain A of A. vinelandii has 480 residues

4To describe the Arg359.N-H...lS2.12 (2.81 A) hydrogen bond ac-curately, the following convention is adopted that allows the dif-ferent nomenclatures that appear in the protein atomic coordinatesand in our quantum description calculations to be used simulta-neously. Second-shell electrostatics-based ligands are derived typi-cally from the X-ray data and the conventional residue label,number and atom designation is assigned, i.e., Arg359.N-H rep-resents the backbone amide of Arg359. First-shell ligands (Fig. 1)are defined by atom type and number, as depicted in the ADFquantum cluster, i.e., lS2.12. The distance from the proton to theappropriate heavy atom is given in parentheses after optimizationof the hydrogen bonding network within the protein (2.81 A)

742

larger effect, but the protein field analysis for these tworesidues suggests the opposite of what might be inferredfrom a simple interpretation of the protein coordinates.

The very weak Lys426.N-H...O.41.HCA (3.90 A) andLys426.Nf1-Hf3...O.41.HCA (3.94 A) hydrogen bond-ing interactions between Lys426 and a single carboxylateoxygen of the homocitrate can be understood in muchthe same fashion as those for Arg359 and Arg96,although the through-space distances are notably longer,and the interaction energy drops off accordingly(–10 kcal/mol). The remaining stabilization associatedwith residues Arg277, Arg361, Arg60 and Arg97 derivespurely due to the favorable electrostatics of positivelycharged residues lying close to a negatively chargedactive site cluster (Fig. 6). These residues are too faraway (>4.7 A) from FeMoco to form even the weakesthydrogen bonding interaction.

Favorable interactions associated with backbone N-H...S type interactions have been noted in the 4Fe4S(Torres RA, Lovell T, Noodleman L, Case DA, manu-script in preparation) and 2Fe2S [42, 61] type proteins.In the 2Fe2S phthalate dioxygenase reductase (PDR)and the Anabaena ferredoxins [42, 61], the number ofpeptide N-H to S hydrogen bonds plays a key role inregulating the redox properties. In PDR, for example,nine peptide dipoles form strong N-H...S bonds witheither the cysteine or inorganic sulfurs of the 2Fe2Scluster. In the Fe protein of nitrogenase from Av, thenumber of N-H...S bonds [to both the 4Fe4S cluster (6)and ligated cysteine thiolates (8)] increases to 14. In thedouble-cubane P cluster of the MoFe protein from Av,there are potentially more than 20 favorable N-H...Sinteractions with the 8Fe7S cluster (10) and cysteinethiolates (12). The increase in the number of interactionsis not unusual and fully in accord with the larger clustersize on going from a 2Fe to a 4Fe to an 8Fe system.

FeMoco is larger in size than the 2Fe2S and 4Fe4Ssystems but approximately equal in size to the P cluster(both the P and M centers contain eight metals,neglecting the homocitrate of FeMoco). However,FeMoco displays only four backbone N-H...S interac-tions around the Mo7Fe9S core (assuming His195 is inthe neutral imidazole state) and two N-H� � �S interac-tions with Cys275. The remaining interactions with theprotein utilize the amino acid sidechains. The dramaticreduction in the number of N-H� � �S interactions asso-ciated with FeMoco (compared to the P cluster) is likelya consequence of FeMoco being the catalytic site ofnitrogenase. In contrast to the electron transferring Pcluster, which is encapsulated by the cage of N-H� � �Sbonds, and therefore held rigidly in the protein, catalysismay require a degree of structural flexibility be given tothe active site by the protein. The direct replacement of anumber of close N-H...S-based interactions by moreflexible, mobile, amino acid sidechains may achieve thisin part. Of the residues involved in N-H...S-basedinteractions outlined in Table 4, the backbone N-Hprotons of Arg359, Leu358, Gly357 and Gly356 (Fig. 7)all lie within hydrogen bonding distance (2.81, 2.62, 2.43

and 2.56 A, respectively) of S12. The summed electro-static contribution of these four residues from thebackbone (–12 kcal/mol) amounts to a value similar tothat observed for the Arg96 sidechain (–13 kcal/mol)and the four N-H...S-type interactions therefore serve asan appropriate substitute for the sidechain.

As noted previously, BS2 is destabilized by the pro-tein environment relative to BS6-3. In order to investi-gate what aspect of the protein environment selectivelydestabilizes BS2, the difference in the protein field terms

Fig. 7. Topological mapping of the amino acid residues thatfeature prominently in the breakdown of the protein fieldinteraction energy onto FeMoco (see text for a detailed discussion).Atoms are identified by color: Fe: magenta; S: yellow; Mo: green; O:red; N: blue; C: dark gray; H: white. The figure was prepared usingMOLSCRIPT [66]

743

(DEp) for states BS6-3 and BS2 is presented in Table 5.In BS6-3, a favorable interaction of the protein back-bone with FeMoco appears to be the primary source ofthe additional stabilization; the negatively charged pro-tein sidechains only make a minor contribution. Theamide protons of Gly356 (–1 kcal/mol) and Gly357(–1 kcal/mol) add to the extra stabilization associatedwith state BS6-3, as does the sidechain of Glu427(–0.81 kcal/mol), but generally the calculated stabiliza-tion of BS6-3 over BS2 in the protein appears to be dueto the cumulative effect of several contributing residuesand no single residue appears to be responsible.

One residue making only a minor contribution(–1.5 kcal/mol) to the protein field, but shown as ahydrogen bonding partner to FeMoco in Fig. 6, isHis195. This residue is often implicated as important forthe delivery of protons to the active site [62]. The pro-tonation state of this residue is currently unknown, butits small contribution from the resulting breakdown ofthe electrostatics is naturally a consequence of the neutralcharge assigned to this residue. This may not be neces-sarily the case for the resting state or for any of the otherreduced or oxidized states of the MoFe protein andFeMoco during turnover, in which case His195 may havea considerable bearing on the resultant electrostaticsprofile shown in Fig. 5. In fact, our calculations suggestthat when the histidine sidechain is in the imidazoliumform, the interaction energy of His195 with FeMoco isabout –11 kcal/mol. However, the protonation states ofthe second-shell amino acid residues of the crystallo-graphically characterized peptide environment are cur-rently unknown and are being investigated via furtherelectrostatics calculations (Lovell T, Asthagiri D, Bash-ford D, Noodleman L, Case DA, unpublished results).

The location of the residues that provide the majorelectrostatic interactions around the active site areshown in Fig. 7. To give a more detailed picture ofspecific positions, three perspectives are shown. The

residues viewed along the three-fold axis from theHis442 end (top) are located in the vicinity of the Mo3Fecubane and are clustered mostly around the lower halfof FeMoco. Those shown from the Cys275 end (bottom)reside more around the 4Fe4S cubane and also surroundthe lower portion of FeMoco. A projection down ontothe top face Fe4Fe5Fe6Fe7 (middle) reveals residues thatsurround the entire central belly of FeMoco exclusivelyand no distinction between upper and lower portions ofFeMoco can be made. The residues outlined in ourelectrostatics profile within hydrogen bonding distanceof the lS2 sulfides (including His195 not shown inFig. 5) are shown in bold.

The most influential destabilizing contributions to theprotein field energy come from the anionic second-shellresidues, Asp228, Glu380, Asp234, Asp386 and Glu427.The total contribution to the protein field of these fiveprotein residues provides 40 kcal/mol destabilization ofthe active site cluster in the protein. All of these residueslie a considerable distance from the FeMoco cluster andthus a more global long-range electrostatics contributionfigures as the likely source of the destabilization asso-ciated with these residues. The contribution of theseanionic residues to the protein field energy is severelydestabilizing. At pH 7, the pKa of an isolated Glu/Aspresidue is 4, suggesting that protonation and a neutralcharge is favorable for these residues. Protonation ofGlu380, for example, results in its protein field destabi-lization energy being reduced from 9 to 3 kcal/mol andoffsets the unfavorable electrostatic interaction with theFeMoco. However, while the pKa of an isolated Glu orAsp residue may be 4, there is an inherent problem withassuming the pKa of Glu380 and all the other Glu andAsp groups in the protein must strictly adhere to a pKa

of 4. The local protein and solvent environment aroundeach residue and the presence of the FeMoco maychange the pKa values of these groups such that theydiffer from what is normally expected. As discussed

Table 5. Difference in the resi-due-by-residue breakdown ofthe protein field interactionenergy (kcal/mol) for the

Mo4+6Fe2+Fe3+ FeMocostates BS6-3 and BS2 dockedinto the 2.0 A A. vinelandiiprotein

Residue DTotal DBack DSide Residue DTotal DBack DSide

Arg359 –0.18 –0.26 0.08 Asp228 0.06 0.00 0.06Arg96 –0.34 –0.07 –0.26 Glu380 –0.53 –0.11 –0.42Lys426 –0.38 –0.82 0.44 Asp234 0.08 –0.02 0.10Arg277 0.76 0.72 0.04 Asp386 –0.18 0.01 –0.19Arg361 0.03 –0.05 0.08 Glu427 –0.80 0.01 –0.81Arg60 –0.26 –0.19 –0.07 Glu440 0.00 0.01 –0.01Arg97 –0.27 –0.13 –0.14 Asp402 0.08 0.00 0.08Lys68 –0.02 0.00 –0.02 Cys62 0.02 0.02 0.00Gly356 –1.05 –1.05 0.00 Asp403 0.01 –0.02 0.03Gly61 –0.90 –0.90 0.00Gly357 –1.11 –1.11 0.00Ile425 0.92 0.92 0.00Leu358 –0.67 –0.67 0.00Cys275 –0.66 –0.66 0.00Total of residues –4.13a –4.28a 0.15a –1.28b –0.10b –1.18b

Total in proteinc –5.41 –2.93 –2.48

aFourteen residues listed above with negative interaction energies are cations or neutralbNine residues listed above with positive interaction energies are anions or neutralcChain A of A. vinelandii has 480 residues. DTotal=(total BS6-3–total BS2). DSide=(side BS6-3–sideBS2). DBack=(back BS6-3–back BS2)

744

above, the FeMoco should shift the pKa of Glu380 from4 to 8.4, but the rest of the protein will also have aneffect, including the titratable sites. Studies in our lab-oratory are now in progress to understand more clearlythe protonation state issues of residues lying close toFeMoco.

Arginines (Arg) appear as a prominent feature of theprotein field and lie close to the lS2 inorganic sulfides,which are formally negatively charged. Arg residues arepositively charged and proton rich and potentially couldact as sources of protons for FeMoco, although theirhigh pKa values must also be considered for activation ofproton transfer by this mechanism. Lying close to theArg residues that surround FeMoco are Asp- and Glu-type residues. These pairs of Arg-Glu and Arg-Asp res-idues counteract each other electrostatically and producea local environment having approximately neutralcharge. The presence of the Asp or Glu type residueslying next to Arg residues also suggests a possible func-tional purpose: the negative charges electrostatically holdthe Arg residues away from the Mo7Fe9S core andthereby prevent the Arg residues forming unfavorablyclose interactions with FeMoco. In this respect, theprotein environment provides the Mo7Fe9S core ampleroom for structural modification during catalysis. Inaddition, given the inherent mobility associated with Aspor Glu sidechains, these negatively charged sidechainsmay be capable of obtaining protons either from bulksolvent [from the 2MIN X-ray data, there is a watermolecule (Wat617) lying very close to one of the lS2

atoms; see Fig. 6] or from close-lying amino acid residues(with low pKa values) and depositing them on FeMoco atpreferred positions. This type of mechanism has beenobserved at the atomic level for 3Fe4S proteins, wherebyfast-proton-transfer kinetics studies have demonstratedthat protons may be captured from water molecules anddeposited on the reduced iron-sulfur cluster core by aflexible Asp sidechain [63]. However, at the MN level, thedistance between the carboxylate sidechains of the Aspand Glu residues and FeMoco is long (5–10 A) and therewould need to be substantial structural rearrangementwithin the first, second and third coordination spheresfor the more reduced turnover states of theMoFe proteinfor this hypothesis to be valid.

Redox interconversion of MOXfiMN

Although not directly observed during enzyme turnover,the addition of redox-active dyes with midpoint poten-tials of ca. 0 to –100 mV to the MoFe protein results in astate in which the S=3/2 EPR signal disappears (MOX).This one-electron oxidation is fully reversible. Moss-bauer [38, 39] and MCD [35, 36, 37] studies haveestablished that MOX has a diamagnetic ground stateand that dav changes by 0.06 mm/s relative to MN [40].

The accurate calculation of the redox potential forFeMoco is made more complicated in Av for severalreasons. The observed S=3/2 EPR signal for MN dis-

appears for the MOX state, indicative of an EPR-silentoxidized state. In principle, the S-based coordinationenvironment around each Fe center suggests it is rea-sonable to assume that each Fe site maintains a high-spinconfiguration and therefore one-electron oxidation ofMN (presumably Fe2+ is the favored oxidation site)should result in either a spin S=1 (total ›–fl elec-trons=2) or spin S=2 (total ›–fl electrons=4) MOX

state. From this simple observation alone it is not obviousthat spin S=0 should be the favored spin state for MOX.5

In addition to the principal difficulty linked to iden-tifying the correct spin structure of MOX, further un-certainty arises due to the most appropriate proteinenvironment that best reflects the MN/POX to MOX/POX

one-electron redox process. The 3MIN structure con-tains metal clusters formally in the MN/PN oxidationstates, while the 2MIN structure has MOX/POX clusters[17]. The transition from 3MIN to 2MIN is associatedwith two successive redox processes, PN to POX (twoelectrons plus one proton redox) and then MN to MOX

(one electron redox). The available protein structurestherefore differ by a total of three electrons and oneproton and there is presently no protein X-ray structuraldata that accurately describes the initial and final statesof the one-electron oxidation process (MN/POX to MOX/POX). Given that the geometry of the FeMoco clusterappears unchanged with little reorganization of the localpolypeptide environment around FeMoco on one-elec-tron oxidation, the 2MIN protein environment is usedfor our MOX to MN redox potential calculations. Themore reduced MN to MR or MI redox potentials aremodeled using the more appropriate 3MIN environ-ment.

In Table 6, using methodology outlined previously[42], calculated redox potentials are presented both inpure solvent (H2O and NMF) and in the protein envi-ronment for the MOX (Mo4+5Fe2+2Fe3+) to MN

(Mo4+6Fe2+Fe3+) interconversion for the three possi-ble different spin states of MOX, S=2, 1 or 0, along withthe measured value of –0.042 V in Av [64]. Redoxpotentials measured in other bacteria are also includedin Table 6 for comparison [15]. Upon one-electron oxi-

dation, regardless of which MOX spin state is examined,

5To obtain the S=0 state for MOX, we assume the spin couplingpattern in BS6-1, that all Fe sites are high spin and the Mo3Fe spintriangle has Fe sites with respective site spins of Fe3(›)=2,Fe4(fl)=–2, Fe5(fl)=–2, giving spin Sa=–2, while the 4Fe¢ cubanehas Fe site spins of Fe6(›)=2, Fe7(›)=2, Fe8(›)=2 and Fe2(fl)=–5/2 to give spin Sb=7/2. The antiferromagnetic coupling of Sa+Sbgives the total spin of the cluster, St=3/2. The removal of oneelectron of minority spin from Fe5 (for example) of the spin triangleyields the S=1 MOX state. The transition of a single electron frombeing minority spin-up on Fe4 to minority spin-down on Fe7 gives astate having an equal number of spin-up and spin-down electrons

and the following site spins: Fe3(›)=2, Fe4(fl)=–5/2, Fe5(fl)=–5/

2, giving spin SaOX=–3, while the 4Fe¢ cubane has Fe site spins of

Fe6(›)=2, Fe7(›)=3/2, Fe8(›)=2 and Fe2(fl)=–5/2 to give

SbOX=3. The important point is that Fe7 (d7) attains an interme-

diate site spin of S=3/2. The antiferromagnetic coupling of

SaOX+Sb

OX=–3+3=0

745

calculated redox potentials differ by almost +0.80 Vfrom the experimental value and appear insensitive tothe choice of spin state used for MOX. Values of around+0.60 V calculated in H2O are closer to the absolutevalue measured in the protein than the calculated valuesin NMF (ca. +0.90 V). From Table 6 and DE re-dox=I.P.+(DEpr)–4.43 [41, 42], the large, positive con-tribution of the environment term (DEpr) outweighs thesmaller gas phase contribution (ca. –2.5 eV) and ensuresthe redox potential is always much higher than thatobserved experimentally. The source of the large envi-ronmental contribution is traced to the change in thereaction field term (DEr�+7.0 eV), which varies in ac-cord with the magnitude of the negative charge associ-ated with FeMoco; the corresponding changes in theprotein field are smaller (DEpr�+0.8 eV) and contributeless to the changing DEpr term.

Nonetheless, the calculated redox potentials are lessaccurate than those calculated previously for other iron-sulfur type proteins ([42], Torres RA, Lovell T, Noo-dleman L, Case DA, manuscript in preparation) andmay be a consequence of the accumulation of severalunknown factors. First, there is a lack of detailedknowledge regarding the spin coupling pattern forMOX, and the precise effect on the geometry of theFeMoco cluster that spin crossings within the Fe man-ifolds will have is not known. Second, the precise pro-tonation states of the FeMoco cluster are not known,particularly with respect to the imidazole sidechain ofHis442 and the carboxylate groups of the homocitrate.Which of the amino acid residues are protonated ordeprotonated in the local peptide environment is alsounresolved. Third, the effect of the protein’s confor-mational change and the associated energetics inresponse to the MOX/POX to MN/POX redox process isundetermined. Finally, there are inherent deficiencies inthe density functional and electrostatics methodology.All of the above represent potential sources of error thatmay surface in the final values of the redox potentialswe have calculated.

One final point which these redox calculations ad-dress concerns the issue of the most appropriate oxida-tion states for the Fe sites at the MN level. Lee et al. [38]proposed an assignment of the metal-ion valencies(Mo4+Fe3+6Fe2+) from their 57Fe Q-band ENDORmeasurements. These valence assignments have recentlybeen challenged, following a revision of the originalMossbauer data analysis [40]. Mo4+4Fe2+3Fe3+ hasbeen proposed, based on a comparison of average iso-mer shift data for FeMoco with that for an Fe2+ modelcomplex with trigonal sulfur coordination. Based onFeMoco cluster charge, size and by direct analogy withthe P cluster of the same protein, we previously sug-gested that if Mo4+4Fe2+3Fe3+ were the correct oxi-dation state for MN, the redox potential for MOXfi MN

would be far more positive than the observed differ-ence of 260 mV compared to the POXfi PN redox po-tential (–300 mV) and also more positive than the

PSUPEROXfi POX couple (+90 mV) [34].Table

6.BreakdownofM

OXto

MNredoxpotentialcalculationsinto

gasphase

ionizationpotentialandinteractionenergyin

theprotein

orin

pure

solvent.aAllenergiesare

given

ineV

MOX

MN

Insolvent

Inprotein

QSpin

Eg

Esol

Ep

Er

Epr

QSpin

Eg

Esol

Ep

Er

Epr

I.P.

DEsol

DE� redox

DEpr

DE� redox

–3

0–429.19

–12.55b

–1.66

–11.46

–13.11

–4

3/2

–426.70

–20.09

–2.54

–18.46

–20.92

–2.49

7.54b

0.62b

7.80

0.88

–12.29c

7.80c

0.88c

–3

1–429.21

–12.54b

–1.68

–11.47

–13.10

–2.52

7.54b

0.55b

7.76

0.82

–12.27c

7.82c

0.89c

–3

2–429.23

–12.50b

–1.72

–11.41

–13.13

–2.53

7.59b

0.63

7.78

0.82

–12.24c

7.85c

0.92c

Experim

ent

–0.32e

0.00f

–0.042g

–0.095h

–0.18i

aM

OX,oxidized

Mcenter;M

N,reducedM

center;Q,charge;Eg,gasphase

energy;I.P.,gasphase

ionizationpotential;Epr,interactionenergyin

theprotein;DE

pr,changein

interaction

energy

intheprotein;Esol,

interaction

energy

inthe

solvent;

DEsol,

changein

the

interaction

energy

inpure

solvent;

DE� redox,redox

potentialcalculated

inaccord

with

DE� redox=

I.P.+

(DEpr)–4.43[41,42]

bCalc.in

H2O

(�=

80)

c Calc.in

NMF(�=

190)

dRef.[15]andreferencestherein

e Ref.[44](inNMF)

f Clostridiumpasteurianum

[64]

gAzotobacterchroococcum

andA.vinelandii[64]

hBacilluspolymyxa[64]

i Klebsiellapneumoniae[64]

746

We have calculated the MOX fi MN redox potentialby assuming that Mo4+4Fe2+3Fe3+ is the correct MN

state. For MOX spin states of 0, 1 and 2, the calculatedredox potentials are +1.36, +1.30 and +1.33 V, re-spectively. Such large shifts towards more positive val-ues for the MOX to MN redox potential support ourprevious conclusion that the Mo4+4Fe2+3Fe3+ oxida-tion level is too oxidized to be the observed MN state[34]. The absolute magnitude of the redox potentialscalculated at the Mo4+6Fe2+Fe3+ level (too positive by+0.80 V) and Mo4+4Fe2+3Fe3+ level (even morepositive by +1.30 V) further suggests that, in order tocalculate accurate redox potentials for FeMoco, a de-tailed working knowledge of several intimately relatedfactors, including the correct spin coupling and oxida-tion state of FeMoco, as well as the protonation state ofFeMoco and the local amino acid environment, are allnecessary.

Redox interconversion of MNfiMR/I

The controlled reduction of the FeMoco in the MoFeprotein has only been successfully achieved for the MOX/POX to MN/POX state; redox potentials for the MN/PN toMR/PN redox couple are not yet available. However,redox data are available for the MOX to MN and MN toMR redox couples for an intact cofactor that has beenextracted in NMF [43, 44, 45, 64] (although the struc-tural integrity of the extracted cofactor is uncertain).Recorded redox potentials for the MOX to MN (–0.32 V)and MN to MR or MI one-electron redox processes(–1.0 V) versus NHE in NMF (�=190 for NMF),respectively, are shown in Table 7, along with our cal-culated values for the MN to MR or MI couples. Themeasured redox potential is observed to vary slightly,depending on the pH of solution used to extract Fe-Moco [43, 44, 45, 64]. Although no measured valuesexist for the MN to MR or MI redox potentials in the

protein, by comparison with the POX to PN couple(–300 mV) the MN to MR or MI redox potential mustbe less than the –300 mV required to reduce POX to PN.If the MN to MR or MI redox potential were greater than–300 mV, the PN cluster would reduce the M center toMR or MI and become PN+. This, however, is in con-tradiction to the observed resting state, which comprisesMNPN and not MRPN+ (where PN+ is one-electronoxidized with respect to PN).

Absolute calculated redox potentials for MN to MR

(+0.15 V) and MN to MI (+0.16 V) are more negativethan the corresponding MOX to MN couple. Values ofMN to MR or MI redox of ca. +0.40 V in H2O areslightly larger; in NMF, values of ca. +0.07 V are cal-culated. While our calculated redox potentials in Table 7for the MN to MR or MI couples differ significantly fromthat reported, the calculated redox potentials for themore reduced couples of MN to MR or MN to MI (ca.+0.16 V in both cases) are more negative than the moreoxidized MOX to MN couple by almost –0.7 V. Experi- T

able

7.BreakdownofM

Nto

MRorM

Iredoxpotentialcalculationsinto

gasphase

ionizationpotentialandinteractionenergyin

theprotein

orin

pure

solvent.aAllenergiesare

given

ineV

MN

MI

Insolvent

Inprotein

QSpin

Eg

Esol

Ep

Er

Epr

QSpin

Eg

Esol

Ep

Er

Epr

I.P.

DEsol

DE� redox

DEpr

DE� redox

–4

3/2

–426.70

–20.09

–2.54

–18.46

–20.92

–5

1–421.34

–30.24b

–3.04

–27.81

–30.85

–5.36

10.15b

0.37b

9.93

0.15

–29.86c

9.77c

0.06c

MR

–5

2–421.42

–30.23b

–3.11

–27.67

–30.79

–5.28

10.14b

0.43b

9.87

0.16

–29.87c

9.78c

0.07c

Experim

ent

–1.00d

<–0.30e

aM

N,oxidized

Mcenter;

MRorM

I ,reducedM

center;Q,charge;Eg,gasphase

energy;I.P.,gasphase

ionizationpotential;Epr,interactionenergyin

theprotein;DE

pr,changein

interactionenergyin

theprotein;Esol,interactionenergyin

thesolvent;DE

sol,changein

theinteractionenergyin

pure

solvent;DE

� redox,redoxpotentialcalculatedin

accord

with

DE� redox=

I.P.+

(DEpr)–4.43[41,42]

bCalc.in

H2O

(�=

80)

cCalc.in

NMF

(�=

190)

dRef.[44](inNMF)

eEstim

atedbasedonPOXto

PNcouple

747

mentally, this difference is £ 0.26 V and in H2O andNMF these calculated differences change to about–0.2 V and –0.8 V, respectively. The shift towards morenegative redox potentials for the more reduced couple inthe protein arises owing to the increased contribution ofthe gas phase ionization potential (I.P.=ca. 5.3 eV) toDE�redox, which offsets the large reaction field contribu-tion (ca. +10.0 eV). Both in the protein environmentand in pure solvents, this qualitative trend in calculatedredox couples, with the MN to MR or MI reduction morenegative than MOX to MN reduction, is consistent withall of the measured redox potentials available for Fe-Moco.

Conclusions

From this energetic analysis, the protein environmentappears capable of the selective stabilization of statesthat would otherwise be considered as higher-lying ex-cited states from calculations done in vacuo. Fromsimple gas phase calculations at the Mo4+6Fe2+Fe3+

level, state BS2 was calculated to be lowest in energy,but BS6 and BS7 were noted to lie within 5 kcal/mol andall 12 BS states were within a 25 kcal range. The closeproximity of several BS states suggested that the relativeenergetic picture in gas phase may change somewhatwhen the effects of the protein and solvent environmentwere included. When the protein and solvent environ-ment effects are switched on, the reverse energetic or-dering is noted for states BS2 and BS6-3, with BS6-3lying lowest. BS6-1 and BS6-2 lie about 2 kcal/molhigher. The BS6 states were previously determined tomatch the experimental data better than any of the otherBS states [34].

For the most favorable BS state, the decompositionof the total protein field energy to individual residueshighlights the importance of the Arg, Lys, Glu and Aspresidues surrounding FeMoco. The Arg359 and Arg96residues are a necessity in that they hydrogen bond tothe cluster; the remainder of the Arg and Lys residuespartially neutralize the large negative charge associatedwith FeMoco and possibly act as proton-rich sources.The favorable electrostatic interactions between Asp-Arg or Glu-Arg pairs of residues also prevents the Argresidues from approaching FeMoco too closely andcreates space around the catalytic FeMoco core. Aspresidues have been noted to participate in mechanismsby which protons undergo relocation via solvent ligandsto 3Fe4S clusters and the prevalence of Asp and Glucarboxylate-type sidechains and water molecules sur-rounding FeMoco may point towards a similar protonabstraction and relocation phenomenon occurring innitrogenase. Ultimately, the effect of mutagenesis ex-periments on the more important residues that we havehighlighted would be most interesting.

While no controlled reduction of the FeMoco to theone-electron reduced state has even been achieved reli-ably in the protein matrix, the available redox data for

the cofactor extracted into organic-based solvents sug-gest that the MN to MR redox potential is more negativethan MOX to MN, as one would expect. Although thecombined density functional and electrostatics calcula-tions do not predict absolute redox data for FeMocowith quantitative accuracy, the calculations do predictthe qualitative trend of a more negative redox potentialfor the more reduced couple. The absolute redoxpotentials calculated are, however, in poorer agreementwith experiment in comparison to redox potentials wehave calculated for ferredoxin iron-sulfur systems ([42],Torres RA, Lovell T, Noodleman L, Case DA, manu-script in preparation).

Several factors need to be investigated more carefullyto account for these quantitative discrepancies. Theseinclude: (1) the protonation states of amino acid residueson or near the FeMoco cluster, which may be unusual insome redox states; (2) the redox potentials are sensitiveto the dielectric model employed, especially in theprotein region, and alternative dielectric models or theadditional use of dynamics calculations have beenproposed [65]; (3) relativistic effects on electronic struc-ture may be significant, particularly for molybdenum(second-row transition element); (4) the protein envi-ronment may significantly polarize the active-sitequantum cluster, which could be assessed with self-consistent reaction field methods or larger quantumclusters; (5) it would be advantageous to have proteinstructures for various redox states and there may beadditional structural uncertainties even in the currentfairly high-resolution structures.

Acknowledgements This work was supported by a NIH grant toD.A.C. (GM 39914). We thank Rhonda Torres, Thomas Rod,Wen-Ge Han, Michael Thompson, Brian Hales, Brian Hoffman,Eckard Munck and Per Siegbahn for useful discussions. We alsothank Duncan McRee for use of the Xtalview progams and EvertBaerends and the Amsterdam group for use of the ADF codes.

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