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Speculative synthetic chemistry and the nitrogenase problem Sonny C. Lee* and Richard H. Holm* Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138 There exist a limited but growing number of biological metal centers whose properties lie conspicuously outside the realm of known inorganic chemistry. The synthetic analogue approach, broadly directed, offers a powerful exploratory tool that can define intrinsic chemical possibilities for these sites while simultaneously expanding the frontiers of fundamental inorganic chemistry. This specula- tive application of analogue study is exemplified here in the evolution of synthetic efforts inspired by the cluster chemistry of bio- logical nitrogen fixation. T he study of synthetic small- molecule analogues is a proven chemical approach for the eluci- dation of biological metal cen- ters. The principal utility of the method was delineated in a definitive early (in 1980) account (1): Synthetic analogues permit (i) the ‘‘deduction of minimal site structure (where unknown) based on coincident model-biomolecule prop- erties or (where known) required for execution of biological function,’’ while further allowing (ii) the ‘‘detection of the influence of the biomolecular envi- ronment on the intrinsic properties of the site as represented by the model.’’ In recent years, the impact of synthetic studies, as construed along these origi- nal, corroborative goals, has diminished due to impressive improvements in bio- logical and biophysical techniques that are allowing the isolation, manipulation, and characterization of biomolecules in ever-increasing speed and detail. More- over, the success of synthetic analogue efforts has rendered most common bio- logical metal motifs well understood at the level of basic inorganic chemistry; issues of deeper biological interest con- cerning these sites are probably unap- proachable through simple synthetic studies. Synthetic analogue chemistry never- theless remains an indispensable compo- nent in the armamentarium of bioinor- ganic investigation. The same advances in biomolecular science that are mitigat- ing the power of corroborative synthetic methods are also revealing the existence of unforeseen, indeed unknown, chemis- try within biological systems. These new frontiers in inorganic and bioinorganic chemistry reside in ‘‘aberrant’’ biological metal centers, sites restricted (appar- ently) to only a few select organisms and distinguished by the presence of unusual metals, peculiar spectroscopic signatures, andor singular chemical re- activity. Where atomic-level definition is available, these centers are marked by surprising complexity that raise but do not answer underlying chemical ques- tions concerning structure and function. Therein lies the third, speculative ob- jective of the synthetic analogue ap- proach: (iii) the broad exploration of fundamental chemistry as motivated by a biological system, with the immediate goal of establishing possibilities and methodologies that ultimately will lead to corroborative synthetic analogues and a chemical understanding of the inspira- tional system. This rationale is not new to synthetic chemistry, but it is growing in prominence because of the obvious gaps in chemical knowledge highlighted by such extraordinary examples as nitro- genase, the photosynthetic water-oxida- tion complex, the hydrogenases, and the carbon monoxide dehydrogenases. We present here a case study of this specu- lative approach as applied to the molec- ular description of nitrogenase. The Synthetic Problem of Nitrogenase Biological nitrogen fixation, the conver- sion of dinitrogen to ammonia by the nitrogenase enzymes (Eq. 1), is the pre- dominant entry point for nitrogen in the global nitrogen cycle (2–7). N 2 8H 8e 16ATP 3 2NH 3 H 2 16ADP 16P i [1] The foundational significance of the process and its intrinsic chemical formi- dability have prompted extensive, ex- tended studies to attain a molecular de- scription of this enzyme system. These efforts have met with limited success, and even simple molecular details such as the minimal requisite reaction stoichi- ometry (Eq. 1 is a limiting, but not nec- essarily obligate formulation), the loca- tion of substrate binding, and the oxidation states of the metals involved remain either unknown or in debate. Few problems in bioinorganic chemistry have proved as challenging and refractory. Progress on this topic is constrained by the complexity of the enzyme and its recalcitrance to standard biochemical and biophysical probes. The system is inaccessible in a poised, ‘‘ready’’ state: Without dinitrogen substrate, the en- zyme spontaneously reduces protons to dihydrogen. Substrate reduction requires a host of other obligatory events (pro- tein dockingrelease, electron transfer storage, ATP hydrolysis, proton transfer, and multisite substrate bindingrelease), leading to a multitude of concurrently populated enzyme states during turn- over and to convoluted kinetics. Enzyme intermediates and inhibited forms are either unobserved or ill-defined. In short, there is no specific information on any active form of nitrogenase. Molecular understanding of this en- zyme therefore is confined to resting- state attributes. We note some salient features: In its best-studied form, the enzyme system consists of two separate soluble components, the Fe protein, the functionally obligate electron donor, and the MoFe protein, responsible for dini- trogen reduction; the MoFe protein con- tains two metallocluster types, the FeMo-cofactor and the P-cluster, both of which are biologically and chemically unprecedented; the site of nitrogen chemistry is inferred (but not absolutely proven!) to occur at the FeMo-cofactor, a complex heterometallic Mo-Fe-S clus- ter, whereas the P-cluster, a high-nucle- arity Fe-S cluster, is thought to mediate electron transfer to the active site; the FeMo-cofactor can be isolated from the native protein as a stable, soluble, but inactive metallocluster that can be rein- corporated into apoprotein with restora- tion of activity; and two related yet ge- netically separate nitrogenase variants are also known that are distinguished by the total absence of molybdenum in both types, the presence of a single va- nadium in the cofactor of one, and the apparent absence of any metal save iron *To whom correspondence should be addressed. E-mail: [email protected] or [email protected]. The distinction between corroborative and speculative studies has been made previously (34). Our present classi- fication subsumes the original conceptions for both cate- gories as largely corroborative while reserving the specu- lative designation for biological situations that demand new chemical perceptions. www.pnas.orgcgidoi10.1073pnas.0630028100 PNAS April 1, 2003 vol. 100 no. 7 3595–3600 SPECIAL FEATURE PERSPECTIVE Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020 Downloaded by guest on March 16, 2020
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Page 1: Speculative synthetic chemistry and the nitrogenase problem · Speculative synthetic chemistry and the nitrogenase problem Sonny C. Lee* and Richard H. Holm* Department of Chemistry

Speculative synthetic chemistry and thenitrogenase problemSonny C. Lee* and Richard H. Holm*Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138

There exist a limited but growing number of biological metal centers whose properties lie conspicuously outside the realm of knowninorganic chemistry. The synthetic analogue approach, broadly directed, offers a powerful exploratory tool that can define intrinsicchemical possibilities for these sites while simultaneously expanding the frontiers of fundamental inorganic chemistry. This specula-tive application of analogue study is exemplified here in the evolution of synthetic efforts inspired by the cluster chemistry of bio-logical nitrogen fixation.

The study of synthetic small-molecule analogues is a provenchemical approach for the eluci-dation of biological metal cen-

ters. The principal utility of the methodwas delineated in a definitive early (in1980) account (1): Synthetic analoguespermit (i) the ‘‘deduction of minimalsite structure (where unknown) basedon coincident model-biomolecule prop-erties or (where known) required forexecution of biological function,’’ whilefurther allowing (ii) the ‘‘detection ofthe influence of the biomolecular envi-ronment on the intrinsic properties ofthe site as represented by the model.’’In recent years, the impact of syntheticstudies, as construed along these origi-nal, corroborative† goals, has diminisheddue to impressive improvements in bio-logical and biophysical techniques thatare allowing the isolation, manipulation,and characterization of biomolecules inever-increasing speed and detail. More-over, the success of synthetic analogueefforts has rendered most common bio-logical metal motifs well understood atthe level of basic inorganic chemistry;issues of deeper biological interest con-cerning these sites are probably unap-proachable through simple syntheticstudies.

Synthetic analogue chemistry never-theless remains an indispensable compo-nent in the armamentarium of bioinor-ganic investigation. The same advancesin biomolecular science that are mitigat-ing the power of corroborative syntheticmethods are also revealing the existenceof unforeseen, indeed unknown, chemis-try within biological systems. These newfrontiers in inorganic and bioinorganicchemistry reside in ‘‘aberrant’’ biologicalmetal centers, sites restricted (appar-ently) to only a few select organismsand distinguished by the presence ofunusual metals, peculiar spectroscopicsignatures, and�or singular chemical re-activity. Where atomic-level definition isavailable, these centers are marked bysurprising complexity that raise but donot answer underlying chemical ques-tions concerning structure and function.

Therein lies the third, speculative† ob-jective of the synthetic analogue ap-proach: (iii) the broad exploration offundamental chemistry as motivated bya biological system, with the immediategoal of establishing possibilities andmethodologies that ultimately will leadto corroborative synthetic analogues anda chemical understanding of the inspira-tional system. This rationale is not newto synthetic chemistry, but it is growingin prominence because of the obviousgaps in chemical knowledge highlightedby such extraordinary examples as nitro-genase, the photosynthetic water-oxida-tion complex, the hydrogenases, and thecarbon monoxide dehydrogenases. Wepresent here a case study of this specu-lative approach as applied to the molec-ular description of nitrogenase.

The Synthetic Problem of NitrogenaseBiological nitrogen fixation, the conver-sion of dinitrogen to ammonia by thenitrogenase enzymes (Eq. 1), is the pre-dominant entry point for nitrogen in theglobal nitrogen cycle (2–7).

N2 � 8H� � 8e� � 16ATP 3 2NH3

� H2 � 16ADP � 16Pi [1]

The foundational significance of theprocess and its intrinsic chemical formi-dability have prompted extensive, ex-tended studies to attain a molecular de-scription of this enzyme system. Theseefforts have met with limited success,and even simple molecular details suchas the minimal requisite reaction stoichi-ometry (Eq. 1 is a limiting, but not nec-essarily obligate formulation), the loca-tion of substrate binding, and theoxidation states of the metals involvedremain either unknown or in debate.Few problems in bioinorganic chemistryhave proved as challenging andrefractory.

Progress on this topic is constrainedby the complexity of the enzyme and itsrecalcitrance to standard biochemicaland biophysical probes. The system isinaccessible in a poised, ‘‘ready’’ state:

Without dinitrogen substrate, the en-zyme spontaneously reduces protons todihydrogen. Substrate reduction requiresa host of other obligatory events (pro-tein docking�release, electron transfer�storage, ATP hydrolysis, proton transfer,and multisite substrate binding�release),leading to a multitude of concurrentlypopulated enzyme states during turn-over and to convoluted kinetics. Enzymeintermediates and inhibited forms areeither unobserved or ill-defined. Inshort, there is no specific informationon any active form of nitrogenase.

Molecular understanding of this en-zyme therefore is confined to resting-state attributes. We note some salientfeatures: In its best-studied form, theenzyme system consists of two separatesoluble components, the Fe protein, thefunctionally obligate electron donor, andthe MoFe protein, responsible for dini-trogen reduction; the MoFe protein con-tains two metallocluster types, theFeMo-cofactor and the P-cluster, bothof which are biologically and chemicallyunprecedented; the site of nitrogenchemistry is inferred (but not absolutelyproven!) to occur at the FeMo-cofactor,a complex heterometallic Mo-Fe-S clus-ter, whereas the P-cluster, a high-nucle-arity Fe-S cluster, is thought to mediateelectron transfer to the active site; theFeMo-cofactor can be isolated from thenative protein as a stable, soluble, butinactive metallocluster that can be rein-corporated into apoprotein with restora-tion of activity; and two related yet ge-netically separate nitrogenase variantsare also known that are distinguished bythe total absence of molybdenum inboth types, the presence of a single va-nadium in the cofactor of one, and theapparent absence of any metal save iron

*To whom correspondence should be addressed. E-mail:[email protected] or [email protected].

†The distinction between corroborative and speculativestudies has been made previously (34). Our present classi-fication subsumes the original conceptions for both cate-gories as largely corroborative while reserving the specu-lative designation for biological situations that demandnew chemical perceptions.

www.pnas.org�cgi�doi�10.1073�pnas.0630028100 PNAS � April 1, 2003 � vol. 100 � no. 7 � 3595–3600

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in the other. Finally, incisive structuraldata on the metalloclusters has becomeavailable, as presented in the followingsections, and this information above allelse has framed the problem for specu-lative synthetic analogue efforts.

X-Ray Absorption Spectroscopy(1978–1992)The first structural insight into theFeMo cofactor originated from Mo-Kedge x-ray absorption spectroscopy ofthe native protein and the isolated co-factor cluster. The extended x-ray ab-sorption fine structure (EXAFS) analy-sis substantiated the existence of aMo-Fe-S cluster, revealing the nearestneighbors of molybdenum as three tofour sulfur atoms at 2.36(2)-Å distanceand two to three iron atoms at 2.72(3)Å (8); the molybdenum environments offree and protein-bound cofactor werefound to be essentially indistinguishableby this technique. Combined with thecompositional data for the FeMo-cofactor (early analyses suggested[MoFe6–8S4–6]), these results offered thefirst reasonable foundation for syntheticwork.

The initial EXAFS results suggested a[MoFe3S3] heterometallic cuboidalstructure as one possible local geometryabout the molybdenum center. Appro-priate synthetic analogues were achievedquickly through self-assembly reactionsemploying [MoS4]2� as both an organiz-ing component and a source of molyb-denum and sulfide (9, 10); a recent,highly efficient variation of this syntheticmethod (11) is illustrated in Scheme 1.The heterocubane clusters thus obtainedpossessed molybdenum environments inexcellent agreement with the originalEXAFS analysis and with subsequentX-ray absorption near edge structure(XANES) studies that indicated a fac-octahedral Mo(IV) coordination spherein the FeMo cofactor consisting of threelight (2p) atom donors and three sulfurs.Later studies would reveal the existenceof an alternate nitrogenase with a vana-dium-containing cofactor; synthetic

[VFe3S4] cubane clusters (12), preparedin a manner similar to the molybdenum-based syntheses, likewise were deter-mined to have a heterometal environ-ment congruent by EXAFS criterion tothat of the biological cluster. To date,these systems are still the most accuratesynthetic structural representations forthe heterometallic portion of their re-spective cofactors. The synthetic devel-opment of [MFe3S4] clusters presaged awider occurrence of the motif in biologi-cal systems, specifically as void-filled‘‘inorganic mutants’’ of 3-Fe sites inferredoxins (13) and, more recently, indistorted form within the C-cluster ofNi-containing carbon monoxide dehy-drogenase (refs. 14 and 15; Fig. 1). Syn-thetic methodology has kept pace withthese developments: In addition to thethiometallate routes used for the earlytransition metal [MFe3S4] clusters, path-ways starting from trinuclear Fe-S clus-ters have allowed access to late metalderivatives as well (Scheme 2), and welldefined, synthetic examples of the[MFe3S4] cubane core are now knownfor M � V, Nb, Mo, W, Re, Co, Ni, Cu,Ag, Tl (13, 16). The accessibility, stabil-ity, and recurrence of the heterometalliccubane motif suggest that additionalbiological examples may surface in thefuture.

The composition of the FeMo-cofac-tor also drew attention to the synthesisof higher-nuclearity Fe-S clusters of sim-ilar stoichiometry. This interest waspredicated on the optimistic notion thatthe nitrogenase cofactor was representa-

tive of some general cluster type of highthermodynamic stability and ready ki-netic accessibility. Both propositionsseemed reasonable at the time, giventhe stability of isolated cofactor and thechemistry of biological Fe-S clusters asunderstood then.‡ Clusters of composi-tional note during this period spanned arange of core structures and terminalligands (ref. 17; Fig. 2). Although mostof these clusters were not directly usefulas synthetic analogues (an unsurprisingoutcome in light of the imprecise analyt-ical data and fragmentary structural in-formation available for the cofactor),they nonetheless contributed tangibly tothe understanding of synthetic Fe-Schemistry. Certain abiological phos-phine-ligated species, however, wouldemerge serendipitously as the progeni-tors for the next generation of cofactor-related synthetic clusters.

The First Macromolecular Structures(1992–2002)The macromolecular structure determi-nation of the MoFe protein (18, 19)marked a watershed in nitrogenasechemistry and in Fe-S biochemistry gen-erally. The diffraction data (2.7-Å reso-lution in 1992) allowed the first directvisualization of the enzyme active site,yielding a structural model for theFeMo-cofactor as a [MoFe7S9] clustercomposed of two sulfur-voided [M4S3]cuboidal fragments linked by three �2-sulfur bridges (Fig. 3). This structurecorroborated the x-ray absorption spec-troscopy predicted local environmentabout molybdenum but also revealed anumber of startling features: six three-coordinate iron sites at the cluster cen-ter, each slightly pyramidalized inward;open, nonrhomb cluster faces aboutthose anomalous iron sites, in contrastto the [Fe2S2] rhombs that typically con-stitute Fe-S cluster geometries; and aseemingly vacant cavity within the clus-ter core. Later, higher-resolution (2.0-Å)protein crystal structures (20) wouldshow the P-cluster to be a symmetric[Fe8S7] structure (Fig. 3) comprising, inthe native state (PN), two [Fe4S3] cuboi-dal halves, vertex-fused through a �6-sulfide and further interconnected bytwo �2-cysteinate bridges; after oxida-tion (the POX state), the cluster wasfound rearranged to a more open, asym-metric geometry, with additional proteinligation provided by a serine side-chainoxygen and a backbone amide nitrogen.All these cluster structures were remark-able from a synthetic perspective, and

‡The prospect of easy synthetic access has waned in recentyears with the discovery of an extensive biosynthetic ap-paratus, external to the nitrogenase enzyme, for cofactorassembly.

Fig. 1. Other [MFe3S4] cubane motifs in biology:void-filled 3-Fe clusters (Left) and the C- cluster in aNi-Fe-S carbon monoxide dehydrogenase (Right).A different structure is available (at lower resolu-tion) for the C-cluster in an analogous enzyme froma different organism that contains the [NiFe3S4]cubane cluster in a less-distorted form (15). M �Mn, Co, Ni, Cu, Zn, Cd, Ga, Tl; N*, histidine; S*,cysteinate.

Scheme 1. Direct synthesis of a single [MoFe3S4]heterometallic cubane cluster.

Scheme 2. Synthesis of a [NiFe3S4] heterometalliccubane cluster.

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new chemistry was clearly needed tounderstand their properties.

As part of synthetic studies that pre-ceded the macromolecular structures,

phosphine-ligated [MFe4S6] (M � Mo,V) species were prepared in a specificattempt to access new (Mo,V)-Fe-Sclusters for comparison with the FeMo-cofactor (Scheme 3; refs. 21 and 22).The biological correspondence wouldturn out not to involve molybdenum atall but rather the remaining Fe4S6 clus-ter fragment, which resembled the co-factor model in possessing a sulfur-voided cuboidal geometry, nonrhombfaces with �2-sulfide bridges, and, forthe phosphine-ligated sites, curious tri-gonal pyramidal metal geometries thatare distributed and flattened (if theaxial ligands are ignored) in a manneranalogous to the three-coordinatesites in the biological cluster. If the[MoFe3S4] cubane core, less one sulfide,can be said to represent one end of theFeMo-cofactor, then the [MFe4S6]core, less the heterometal, would consti-tute the remainder. The idiosyncratic,cofactor-like features of the [MFe4S6]clusters (i.e., trigonal pyramidal metalsites and nonrhomb faces) are discern-ible elsewhere only in the [Fe6,7S6]-X-PR3 clusters shown in Fig. 2 (21); aphosphine ligand environment thereforesurfaced as the common denominatorfor these structural features, perhapsdue to the preferential stabilization ofreduced oxidation states by this ligand

class relative to anion donors. This per-ceived relationship, conjoined with spec-ulation that the FeMo-cofactor might bea predominantly ferrous cluster in theinactive, structurally characterized state,encouraged deeper investigation intophosphine-ligated Fe-S species.

Reduction of phosphine-ligated[MFe3S4] cubanes (M � Fe, Mo, V)yielded edge-bridged double-cubane[M2Fe6S8] clusters that approach thecore composition and overall extendedgeometry (as opposed to more compact,higher-symmetry structures) of symme-trized versions of the FeMo-cofactor(23–25). In an effort to realize clusterswith cofactor-exact 8:9 metal�sulfidestoichiometries, reactions of thesedouble-cubane products with sulfide re-agents were explored; we note two rele-vant discoveries. First, treatment of a[Mo2Fe6S8] cluster with two equivalentsof (Et4N)SH resulted not in insertion ofsulfide ligands but rather the isolation ofa reduced cluster of unaltered compo-sition but rearranged core geometry(Scheme 4, a; ref. 26). This rearrange-ment was notable in the emergence oftight metal–metal interactions that giverise to ostensibly three-coordinate ironsites (if an FeOFe bond is excludedfrom consideration); this represents theclosest approach thus far to a three-coordinate iron-sulfide environment insynthetic chemistry. Second, if relatedbut more reduced [M2Fe6S8] clusters arereacted with three to four equivalents ofthe same hydrosulfide reagent, net in-corporation of sulfur is achieved, withformation of rearranged cluster cores ofthe desired [M8S9] composition (Scheme4, b; ref. 27). Despite the constitutionalsimilarity, the resultant clusters do notadopt cofactor-like cores but insteadbear a striking near-congruence to thenative form of the nitrogenase P-cluster(if bridging �2-sulfides are substitutedfor the �2-cysteinates in the biologicalsystem). This structural motif also oc-curs in other, more complex clustersderived from sulfidation of reducededge-bridged double-cubane species (27)and therefore seems to reflect intrinsi-cally accessible and stable chemistry. If

Fig. 2. Selected higher-nuclearity Fe-S clusters. (Y, z) � (N2H4, 4), (OH�N3�CN, 5), (S, 6); X, monoanions(halide�thiolate�aryloxide).

Fig. 3. Metalloclusters in the nitrogenase MoFeprotein as derived from protein crystallography(pre-2002). N*, histidine; O*, serine�serinate; S*,cysteinate.

Scheme 3. Synthesis of [MFe4S6] heterometallicclusters. (M, x) � (Mo, 4), (V, 7).

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this sulfide-induced rearrangement isgeneral for juxtaposed, reduced Fe-Scubane clusters, this transformationcould represent the chemical basis bywhich the P-cluster is assembled. Theexistence of substantive redox- andsulfide-dependent rearrangements inextended, higher-nuclearity clusters inboth synthetic and biological (i.e., POX

vs. PN) contexts also introduces otherramifications. First, the prospect of clus-ter rearrangement suggests a possiblerole during the function of the FeMo-cofactor; this is discussed further in thenext section. Second, the compositionaland geometric similarities between theFeMo-cofactor and PN-cluster structures(both are octanuclear with cuboidalhalves) and their occurrence within thesame protein hint at a chemical andevolutionary relationship by core rear-rangement. These are intriguing proposi-tions for further study.

A different synthetic approach em-phasizes the construction of the individ-ual sulfur-voided cuboidal subunitswithin the FeMo-cofactor. The isolated[Fe4S3] core is a long-known but exceed-ingly rare Fe-S motif, and examples arelimited to the venerable homoleptic ni-trosyl cluster known as Roussin’s blackanion {[Fe4S3(NO)7]�} and to phos-phine derivatives of more recent vintage{[Fe4S3(NO)4(PR3)3]0,�} (28). Coucou-vanis et al. (29) have prepared analo-gous heterometallic [MoFe3S3] clustersthat resemble the other subunit of thecofactor by carbonylation of an edge-bridged double-cubane cluster (Scheme4, c); this reaction presumably proceeds

by formation of a CO-ligated single-cubane species followed by phosphine-coupled desulfurization. From these fewexamples, it seems at present thatstrong-field, low-valent (subferrous) ironsites are necessary to stabilize [MFe3S3]cuboidal cores in discrete form.

The Interstitial Atom (2002)Very recently, a high-resolution crystal-lographic analysis (1.16 Å) of the MoFeprotein has revealed a �6-interstitialatom§ at the center of the FeMo-cofac-tor (Fig. 4; ref. 30). This discovery, ifcorrect, is arguably the most significantaddition to nitrogenase knowledge sincethe advent of the first macromolecularstructures. Although the new structurerefinement did not allow identificationof this ligand with certainty, the electrondensity and bond distances are consis-tent with a light (2p) element, perhapscarbon, nitrogen, or oxygen, and theresolution dependence of the electrondensity best matches the curve gener-ated by a nitrogen atom. These consid-erations plus the universal acceptance ofthe FeMo-cofactor as the site of nitro-gen reduction have led to the tentativeassignment of nitride as the newfoundcore ligand.

The most obvious consequence of thisfinding stems directly from its impact oncluster environment: The revelation ofthe internal ligand makes all iron cen-

ters in the cofactor four-coordinate and(distorted) tetrahedral. This revisioneliminates the most chemically troublingaspect of the original structural model,the bizarre three-coordinate, unsatur-ated iron sites that have remained no-ticeably absent in synthetic systems, andit removes any need to invoke arcaneexplanations such as metal–metal bond-ing, very reduced iron states, or indis-cernible hydride ligands to account forthe resting-state cluster geometry. Al-though the FeMo-cofactor is still singu-lar and remarkable from an inorganicperspective, it can now be viewed (reas-suringly) as a structurally and chemicallyplausible, albeit unusual, weak-field Fe-Scluster.

The existence of this central atomraises related questions of provenanceand function. Does this ligand originatefrom cluster biosynthesis or cluster reac-tivity? If the former, does this ligandhave some function as an organizing ortemplating agent during cluster assem-bly? If the latter, does the internal li-gand represent adventitious trapped ma-terial (nitride from substrate, oxidefrom water?) within a dead-end clusterform outside the catalytic cycle, or is itan essential participant in or mediatorof cluster function? In keeping with thenature of this subject, the addition of asingle atom has raised far more ques-tions than answers.

The structural revision of the FeMo-cofactor nevertheless offers a cautionarymechanistic tale. In the original cofactormodel, the presence of six putativethree-coordinate iron sites, togetherwith the apparent existence of an all-Fenitrogenase, led to wide speculation(ref. 31 and references cited therein)concerning the participation of iron indinitrogen reduction and, in some con-jectures, the consequent incorporationof nitrogen ligation as part of the Fe-Scluster core during catalysis. Theamended cofactor model now containsonly coordinatively stable metal centersand no obvious substrate binding sites.There is an irony here: The addition ofthe interstitial donor removes much ofthe impetus behind Fe-mediated mecha-nistic hypotheses, yet if the ligand turns

§The original void in the cofactor core now seems to be anartifact of Fourier-series termination effects arising fromthe limited resolution of the earlier data.

Fig. 4. Revised FeMo-cofactor structure derivedfrom the most recent, high-resolution macromo-lecular structure determination. N*, histidine; S*,cysteinate.

Scheme 4. Selected reactions of edge-bridged double-cubane clusters. LnM � (Et3P)(Cl4cat)Mo forCl4cat-ligated products or TpM for Tp-ligated products; Cl4cat, tetrachlorocatecholate(2�); Tp, hydro-tris(pyrazolyl)borate(1�); (M, x � z) � (Mo, 3), (V, 4).

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out to be nitride, it could also providethe first (and only) direct observationalevidence¶ to support those original pro-posals! The seeming absence of a bind-ing site in the resting-state cofactorstructure suggests that structuralchanges are needed before the clusterbecomes substrate-competent. If the in-terstitial ligand actually participates inthe reaction chemistry, either directlyduring turnover or indirectly by activa-tion�release before catalysis, dramaticrearrangements of the cluster core ge-ometry will certainly occur. We havenoted the observation of major redox-and chemically induced rearrangementsin synthetic clusters of roughly compara-ble composition and geometry. The im-plications raised by the new interstitialligand and the synthetic chemistry un-derscore the hazards in formulatingmechanistic hypotheses based onresting-state perceptions.

New Synthetic FrontiersThe structural revision of the FeMo-cofactor necessitates a reappraisal ofsynthetic strategy. The focus of immedi-ate attention is, of course, the new mon-atomic ligand. What is its identity? Thisis the overriding first concern, becausethe chemistries of carbide, nitride, andoxide (if these are indeed the only can-didates) differ radically. Although thisissue is currently unsettled, we will as-sume it to be nitride for the presentdiscussion.

In molecular transition metal chemis-try,� nitride is found most commonly asa terminal ligand (�300 structurallycharacterized complexes); to sustain its3� formal charge at a single metal site,a multiply bonded metal-nitride moietyand a high-valent metal are practicalcriteria, and known examples are re-stricted to groups 5–8. As bridging li-gands, nitrides have been observed tospan anywhere from two to six metalcenters, with dinuclear bridges beingmost prevalent (�150 complexes). For�2-nitrides, multiple bonding remainsimportant, although the lower per-metalbond order and greater charge delocal-ization expand the range of acceptableenvironments relative to the terminalbinding mode. In addition, localizationof the multiple bond and asymmetricbridging are widespread in these sys-tems, particularly for mixed [M(�2-

N)M�] units; very disparate local metalenvironments therefore are possiblewithin the dinuclear fragment as long asone site is an early-to-middle transitionelement in a moderate-to-high oxidationstate.

Of greatest interest in the presentcontext are cluster-bound nitrides, i.e.,bridging nitrides of �3 or higher, whichexist within a smaller set of compounds(�100 total, about evenly distributed for�3–�6 cases). These nitride complexesfall neatly into three general classes:(i) mid- to high-valent early-transitionmetal clusters (groups 4–6), which de-scribe all complexes containing �3-nitrides and can be viewed as a furtherextension of the environment describedfor terminal and �2 species; (ii) low-valent middle- to late-transition metalcarbonyl clusters (mainly groups 8–9 butalso heterometallic cores containingthese elements plus group 6–10 metals),in which almost all nitrides with thehighest bridging modes (�4–6) occur;and (iii) Au(I) phosphine clusters, whichform a meager set of �4- and �5-N com-pounds distinguished by their uniqueaurophilic structures. To some extent,the preference of oxidized early metalclusters for lower nitride-bridging modescan be attributed to their electron defi-ciency, which makes � donation fromthe nitride bridge more favorable thanadditional �-derived bridging.** For the

reduced, electron-rich metal centers inthe carbonyl and gold clusters, addi-tional bridging seems advantageous incompensating for the basicity of the ni-tride ligand.

The various nitride-bridging modesmanifest regular preferences for coordi-nation geometry (Table 1). True inter-stitial nitrides, as defined by completelyinternal coordination within a clusterpolyhedron, are encountered at �4,5 forAu(I) and �5,6 for transition metal clus-ters. From the existing examples, bridg-ing modes in excess of �6 seem unfavor-able for nitride; in cases where thepotential interstitial nuclearity can ex-ceed six, the internal nitrides are invari-ably found coordinated to only a pen-tanuclear (�5) subset.

It is clear from our brief survey thatthe environment in the FeMo-cofactoris without direct precedent in nitridechemistry. The dissimilarities arise notfrom simple differences in ligand set orcluster geometry but rather from funda-mental divergences in essential metalproperties. For example, iron nitridesare known in tetra-, penta-, and hex-anuclear carbonyl clusters (Fig. 5) andin oxidized dinuclear species supportedby multidentate ancillary ligands (32).Likewise, the trigonal prismatic �6-Ninterstitial geometry occurs in metal car-bonyl clusters (see Table 1). Weak-field,exchange-labile, redox-active environ-ments typical of the iron sites in Fe-Sclusters, however, do not exist in nitridechemistry as it currently stands. The de-velopment and adaptation of new andexisting fundamental nitride chemistryto this end is therefore a clear and

¶There is also a strangely appropriate correspondence toindustrial nitrogen fixation (the Haber–Bosch process),where iron-coordinated, surface-bound nitrides are thefirst chemical intermediates observed after dissociativechemisorption of dinitrogen (see ref. 35).

�Our analysis is based on a survey of the Cambridge Struc-tural Database, version 5.23 (April 2002) (Cambridge Uni-versity, Cambridge, England).

**This correlation is not absolute, as demonstrated by iso-lated instances of �5- and �6-nitride in penta- and hexa-nuclear amidoimidonitrido Zr(IV) clusters (CambridgeStructural Database refcodes VUHHIP and VUHHOV) (see� footnote).

Table 1. Cluster-bridging nitride geometries and representative complexes

Bridging geometry Representative cluster CSD refcode*

�3

Pyramidal [{Cp*Ti(�-NH)}3(�3-N)] SAXSEPT-shaped [{CpMo(CO)2}2{CpMo(O)}(�3-N)] BAXFELTrigonal planar† [{CpMo(O)(SMe)(�3-

N)}{(CpMo)2(�2-SMe)3}]GOTZO

�4

Sawhorse [Fe4(�4-N)(CO)12]� PRUFEBBasal‡ trigonal pyramidal† [PtRu3(�-H)(�4-N)(CO)10P(i-Pr)3] QUDMORInterstitial tetrahedral§ [Au4(�4-N)(PPh3)4]� CUGPUP

�5

Basal‡ square pyramidal [Fe5(�5-N)(�-CO)2(CO)12]� CABROM10Interstitial distorted¶ [PtRh10(�5-N)(�-CO)10(CO)11]3� BENPOZInterstitial trigonal bipyramidal§ [Au5(�5-N)(PPh3)5]2� SEVJUY

�6

Interstitial octahedral [Fe6(�6-N)(�-CO)3(CO)12]3� ZUCWAVInterstitial trigonal prismatic [Co6(�6-N)(�-CO)9(CO)6]� PIMNCO

*Cambridge Structural Database, version 5.23.†Rare.‡Exposed nitrides centered in (or protruding slightly from) the basal face (triangular or square) of apyramidal coordination polyhedron.

§Au(I) only.¶Usually found within high-nuclearity clusters.

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present synthetic challenge that must beaddressed in conjunction with (and per-haps before) efforts aimed at the sys-tematic preparation of (Mo-)Fe-S-Nclusters. We note that the recent chem-istry of iron-amide and -imide clusters(33) may have some bearing on thistopic. These clusters (Fig. 6) are ligatedby basic nitrogen anion donors that arechemical cousins to nitride, evince prop-erties that broadly resemble those ofFe-S clusters, and, in terms of metalattributes, are perhaps more closely con-nected to hypothetical cofactor-relevantnitride environments than existing iron-nitride species. Their synthesis and reac-tivity characteristics therefore could pro-vide guidance and possible startingpoints for further investigation directedtoward weak-field iron-nitride chemistry.

ProspectsTraditionally, synthetic inorganic chem-istry has provided the molecular intu-ition needed to interpret metal behavior

in biological systems. Today, in a rever-sal of circumstance, metallobiomoleculesare posing fundamental inorganic ques-tions whose answers lie outside our ex-isting knowledge. It is a fitting symmetrythat the relationship between inorganicchemistry and biology has come fullcircle.

Speculative synthetic analogues canplay a critical role in bridging the twodomains and advancing both. The con-tinuing synthetic investigation of nitro-genase illustrates the synergies of thisapproach. As described in this account(and we have not discussed the exten-sive enzyme-inspired studies aimed atfunctional simulation), the speculativeapproaches motivated by this demand-ing problem have provided insight intothe metalloclusters of nitrogenase.Equally important, these efforts havealso contributed broadly to basic areasof inorganic chemistry that wouldprobably not have been examined oth-erwise; the intrinsic value of such ex-

ploration should not be underesti-mated. The most recent revision of theFeMo-cofactor structure presents yetanother opportunity to expand our un-derstanding of the biological system inparticular and of fundamental inor-ganic chemistry in general.

The synthetic problem of nitrogenase,nevertheless, remains unsolved. To thiswe give Captain Ahab’s oath: ‘‘Aye, aye!and I’ll chase him round Good Hope,and round the Horn, and round theNorway Maelstrom, and round perdi-tion’s f lames before I give him up.’’

We thank Dr. R. Panda for assistance. Re-search on the subject of this perspective hasbeen supported by National Institutes ofHealth Grant GM-28856 (to R.H.H.), Na-tional Science Foundation Grant CHE-9984645 (to S.C.L.), and an Arnold and Ma-bel Beckman Foundation Young InvestigatorAward (to S.C.L.).

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2. Howard, J. B. & Rees, D. C. (1996) Chem. Rev.(Washington, D.C.) 96, 2965–2982.

3. Burgess, B. K. & Lowe, D. J. (1996) Chem. Rev.(Washington, D.C.) 96, 2983–3011.

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9. Wolff, T. E., Berg, J. M., Hodgson, K. O., Frankel,R. B. & Holm, R. H. (1979) J. Am. Chem. Soc. 101,4140–4150.

10. Wolff, T. E., Berg, J. M., Power, P. P., Hodgson,K. O. & Holm, R. H. (1980) Inorg. Chem. 19,430–437.

11. Fomitchev, D. V., McLauchlan, C. C. & Holm,R. H. (2002) Inorg. Chem. 41, 958–966.

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13. Johnson, M. K., Duderstadt, R. E. & Duin, E. C.(1999) Adv. Inorg. Chem. 47, 1–82.

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15. Drennan, C. L., Heo, J., Sintchak, M. D., Schre-iter, E. & Ludden, P. W. (2001) Proc. Natl. Acad.Sci. USA 98, 11973–11978.

16. Zhou, J., Raebiger, J. W., Crawford, C. A. &Holm, R. H. (1997) J. Am. Chem. Soc. 119, 6242–6250.

17. Dance, I. & Fisher, K. (1994) Prog. Inorg. Chem.41, 637–803.

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20. Peters, J. W., Stowell, M. H. B., Soltis, S. M.,Finnegan, M. G., Johnson, M. K. & Rees, D. C.(1997) Biochemistry 36, 1181–1187.

21. Nordlander, E., Lee, S. C., Cen, W., Wu, Z. Y., Na-toli, C. R., Di Cicco, A., Filipponi, A., Hedman, B.,Hodgson, K. O. & Holm, R. H. (1993) J. Am.Chem. Soc. 115, 5549–5558.

22. Cen, W., MacDonnell, F. M., Scott, M. J. & Holm,R. H. (1994) Inorg. Chem. 33, 5809–5818.

23. Zhou, H.-C. & Holm, R. H. (2003) Inorg. Chem.42, 11–21.

24. Demadis, K. D., Campana, C. F. & Coucouvanis,D. (1995) J. Am. Chem. Soc. 117, 7832–7833.

25. Hauser, C., Bill, E. & Holm, R. H. (2002) Inorg.Chem. 41, 1615–1624.

26. Osterloh, F., Achim, C. & Holm, R. H.(2001) Inorg. Chem. 40, 224–232.

27. Zhang, Y., Zuo, J.-L., Zhou, H.-C. & Holm, R. H.(2002) J. Am. Chem. Soc. 124, 14292–14293.

28. Goh, C. & Holm, R. H. (1998) Inorg. Chim. Acta270, 46–54.

29. Coucouvanis, D., Han, J. & Moon, N. (2002)J. Am. Chem. Soc. 124, 216–224.

30. Einsle, O., Tezcan, F. A., Andrade, S., Schmid, B.,Yoshida, M., Howard, J. B. & Rees, D. C. (2002)Science 297, 1696–1700.

31. Lovell, T., Li, J., Case, D. A. & Noodleman, L.(2002) J. Am. Chem. Soc. 124, 4545–4547.

32. Dutta, S. K., Beckmann, U., Bill, E., Weyhermul-ler, T. & Wieghardt, K. (2000) Inorg. Chem. 39,3355–3364.

33. Duncan, J. S., Nazif, T. M., Verma, A. K. & Lee,S. C. (2003) Inorg. Chem. 42, 1211–1224.

34. Hill, H. A. O. (1976) Chem. Br. 12, 119–123.35. Schlogl, R. (1997) in Handbook of Heterogeneous

Catalysis, eds. Ertl, G., Knozinger, H. & Weit-kamp, J. (VCH, New York), Vol. 4, pp. 1697–1748.

Fig. 5. Representative nitride-containing iron-carbonyl clusters: [Fe4(�4-N)(CO)12]� (Left), [Fe5(�5-N)(�-CO)2(CO)12]� (Center), and [Fe6(�6-N)(�-CO)3(CO)12]3� (Right). See Table 1 for corresponding CambridgeStructural Database refcodes.

Fig. 6. Representative weak-field iron-imide clus-ters. X, monoanions (halide�thiolate).

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Corrections

SPECIAL FEATURE, PERSPECTIVE. For the article ‘‘Speculative syn-thetic chemistry and the nitrogenase problem,’’ by Sonny C. Leeand Richard H. Holm, which appeared in issue 7, April 1, 2003,of Proc. Natl. Acad. Sci. USA (100, 3595–3600; first publishedMarch 17, 2003; 10.1073�pnas.0630028100), the figures andschemes should have been published in color. The correctedillustrations and their legends appear below.

Fig. 2. Selected higher-nuclearity Fe-S clusters. (Y, z) � (N2H4, 4), (OH�N3�CN, 5), (S, 6); X, monoanions (halide�thiolate�aryloxide).

Fig. 1. Other [MFe3S4] cubane motifs in biology: void-filled 3-Fe clusters(Left) and the C-cluster in a Ni-Fe-S carbon monoxide dehydrogenase (Right).A different structure is available (at lower resolution) for the C-cluster in ananalogous enzyme from a different organism that contains the [NiFe3S4]cubane cluster in a less-distorted form (15). M � Mn, Co, Ni, Cu, Zn, Cd, Ga, Tl;N*, histidine; S*, cysteinate.

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Fig. 3. Metalloclusters in the nitrogenase MoFe protein as derived fromprotein crystallography (pre-2002). N*, histidine; O*, serine�serinate; S*,cysteinate.

Fig. 4. Revised FeMo-cofactor structure derived from the most recent,high-resolution macromolecular structure determination. N*, histidine; S*,cysteinate.

Fig. 5. Representative nitride-containing iron-carbonyl clusters: [Fe4(�4-N)(CO)12]� (Left), [Fe5(�5-N)(�-CO)2(CO)12]� (Center), and [Fe6(�6-N)(�-CO)3(CO)12]3�

(Right). See Table 1 for corresponding Cambridge Structural Database refcodes.

Fig. 6. Representative weak-field iron-imide clusters. X, monoanions(halide�thiolate).

Scheme 1. Direct synthesis of a single [MoFe3S4] heterometallic cubanecluster.

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Scheme 2. Synthesis of a [NiFe3S4] heterometallic cubane cluster.

Scheme 3. Synthesis of [MFe4S6] heterometallic clusters. (M, x) � (Mo, 4),(V, 7).

Scheme 4. Selected reactions of edge-bridged double-cubane clusters. LnM � (Et3P)(Cl4cat)Mo for Cl4cat-ligated products or TpM for Tp-ligated products;Cl4cat, tetrachlorocatecholate(2�); Tp, hydrotris(pyrazolyl)borate(1�); (M, x � z) � (Mo, 3), (V, 4).

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ECOLOGY. For the article ‘‘Rapid loss of genetically based resis-tance to metals after the cleanup of a Superfund site,’’ by JeffreyS. Levinton, E. Suatoni, William Wallace, Ruth Junkins, Bren-dan Kelaher, and Bengt J. Allen, which appeared in issue 17,August 19, 2003, of Proc. Natl. Acad. Sci. USA (100, 9889–9891;first published August 6, 2003; 10.1073/pnas.1731446100), sev-eral green squares representing South Cove were missing fromFig. 1B due to a printer’s error. The corrected figure and itslegend appear below.

Fig. 1. Loss of resistance of the oligochaete L. hoffmeisteri after a Cdcleanup. (A) Comparison of mortality curves for Foundry Cove and South CoveL. hoffmeisteri in 1993, just before the cleanup, and in August 2002. (B)Change in time to 50% mortality after Cd exposure for Foundry Cove and forunpolluted South Cove, before and after the cleanup. Lines show model Iregression best fits of trends in change of resistance over time. Probabilities forANOVA of regression are also shown. (C) Cd concentrations of Foundry CoveL. hoffmeisteri in 1984 and in 2002, 8 yr after the cleanup, in Foundry Cove andSouth Cove.

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GENETICS. For the article ‘‘Minimal haplotype tagging,’’ by PaolaSebastiani, Ross Lazarus, Scott T. Weiss, Louis M. Kunkel, IsaacS. Kohane, and Marco F. Ramoni, which appeared in issue 17,August 19, 2003, of Proc. Natl. Acad. Sci. USA (100, 9900–9905; firstpublished August 4, 2003; 10.1073/pnas.1633613100), the authorsnote that Table 1 was printed incorrectly. The following genesshould have listed a number 5 instead of 6: On page 9902, under

African American, the genes CRF, FGB, IL20, and IL3 in thecolumn labeled htSNPs, and under European American, the genesBDKRB2, DEFB1, and IL24 in the column labeled htSNPs; and onpage 9903, under African American, the gene PLAUR in thecolumn labeled Haplotypes, the genes TLR3, TLR4, and TLR7 inthe column labeled htSNPs, and the gene SERPINE1 in the columnlabeled Time, sec. The corrected table appears below.

Table 1. Results of the analysis of 105 genes using BEST

Gene SNPs

African American European American Shared SNPs

Haplotypes htSNPs Ratio, % Time, sec Haplotypes htSNPs Ratio, % Time, sec Number Ratio, %

ACE2 57 13 7 12 0 12 6 11 1 6 100BDKRB2 28 12 8 29 1 7 5 18 0 5 100BPI 35 9 5 14 0 9 5 14 0 5 100CARD15 19 6 4 21 0 4 2 11 0 2 100CCR2 23 10 7 30 0 6 3 13 0 3 100CEBPB 8 5 4 50 0 2 1 13 0 1 100CLCA1 103 3 2 2 0 3 2 2 0 2 100CRF 21 10 5 24 0 8 4 19 0 4 100CRP 18 10 6 33 0 9 6 33 0 6 100CSF2 14 11 6 43 0 8 4 29 0 4 100CSF3 12 6 5 42 0 2 1 8 0 1 100CSF3R 41 14 6 15 0 11 5 12 5 5 100CYP4F2 79 10 5 6 23 8 4 5 1 4 100DCN 66 3 2 3 0 3 2 3 0 2 100DEFB1 85 11 6 7 52 9 5 6 4 5 100F11 69 10 5 7 8 9 4 6 2 4 100F2 31 7 5 16 0 7 5 16 0 5 100F2R 42 8 4 10 0 8 4 10 0 4 100F2RL1 29 7 4 14 0 7 4 14 0 4 100F2RL2 26 13 6 23 0 10 5 19 0 5 100F2RL3 23 9 7 30 0 7 5 22 0 5 100F3 22 10 6 27 0 7 4 18 0 4 100F7 20 8 5 25 0 5 3 15 0 3 100F9 51 12 7 14 0 10 4 8 1 3 75FGA 8 7 5 63 0 2 1 13 0 1 100FGB 29 6 5 17 0 3 2 7 0 2 100FGG 8 6 4 50 0 3 2 25 0 2 100FGL2 10 6 5 50 0 3 2 20 0 2 100FSBP 17 6 5 29 0 3 2 12 0 2 100GP1BA 13 8 6 46 0 3 2 15 0 2 100IFNG 8 7 5 63 0 3 2 25 0 2 100IGF2 13 11 7 54 0 7 4 31 0 4 100IL10 19 8 7 37 0 2 1 5 0 1 100IL11 23 12 6 26 0 10 5 22 0 5 100IL12A 26 11 9 35 0 8 6 23 0 6 100IL12B 25 8 6 24 0 4 3 12 0 3 100IL13 18 11 7 39 0 10 6 33 0 6 100IL17B 16 5 4 25 0 3 2 13 0 2 100IL18 41 9 6 15 0 6 5 12 0 4 80IL18BP 8 6 4 50 0 4 3 38 0 3 100IL19 19 7 5 26 0 4 3 16 0 3 100IL1B 24 9 5 21 0 7 4 17 0 4 100IL1R2 97 7 4 4 0 5 3 3 0 3 100IL2 7 6 5 71 0 3 2 29 0 2 100IL20 9 11 5 56 0 8 4 44 0 4 100IL21R 45 6 4 9 0 6 4 9 0 4 100IL22 21 9 4 19 1 6 3 14 0 3 100IL24 19 9 6 32 0 6 5 26 0 5 100IL3 7 8 5 71 0 5 3 43 0 3 100IL4 50 4 3 6 0 4 3 6 0 3 100IL5 5 7 5 100 0 5 3 60 0 3 100IL6 21 12 9 43 0 10 7 33 0 7 100IL8 7 7 5 71 0 5 3 43 0 3 100IL9 8 6 4 50 0 3 2 25 0 2 100

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Table 1. Continued

Gene SNPs

African American European American Shared SNPs

Haplotypes htSNPs Ratio, % Time, sec Haplotypes htSNPs Ratio, % Time, sec Number Ratio, %

IL9R 51 5 3 6 0 5 3 6 0 3 100ITGA2 229 2 1 0 0 2 1 0 0 1 100JAK3 60 4 2 3 0 3 2 3 0 1 50KEL 58 6 4 7 0 4 2 3 0 2 100KLK1 35 6 4 11 0 6 4 11 0 4 100LBP 37 8 5 14 0 6 3 8 0 3 100LTB 5 6 4 80 0 4 2 40 0 2 100LY64 40 10 6 15 0 9 5 13 0 5 100MC1R 19 7 5 26 0 5 3 16 0 3 100MD-1 12 8 5 42 0 5 3 25 0 3 100MD-2 9 6 3 33 0 3 2 22 0 2 100MMP3 22 14 7 32 0 14 7 32 0 7 100NOS3 43 9 5 12 0 9 5 12 0 5 100PLAU 18 7 6 33 0 4 3 17 0 3 100PLAUR 65 5 3 6 0 5 3 5 0 3 100PLG 106 9 5 5 1 9 5 5 1 5 100PON1 103 8 3 3 2 8 3 3 2 3 100PPARA 64 3 2 3 0 2 1 2 0 1 100PPARG 84 11 5 6 13 11 5 6 13 5 100PROC 29 11 5 17 0 8 4 14 0 4 100PROZ 35 7 4 11 0 6 3 9 0 3 100SCYA2 23 9 6 26 0 4 3 13 0 3 100SELE 46 10 7 15 0 6 4 9 0 4 100SELP 96 4 2 2 0 4 2 2 0 2 100SERPINA5 40 11 6 15 0 6 4 10 0 3 75SERPINC1 23 10 8 35 0 7 6 26 0 5 83SERPINE1 40 10 7 18 5 8 5 13 0 5 100SFTPB 25 13 6 24 1 11 5 20 1 4 80SFTPD 87 7 4 5 0 7 4 5 0 4 100SMP1 39 10 6 15 0 8 4 10 0 4 100STAT4 37 10 5 14 7 8 5 14 3 5 100STAT6 19 12 5 26 0 11 5 26 0 5 100TGFB3 37 8 5 14 0 6 4 11 0 4 100THBD 6 7 4 67 0 3 2 33 0 2 100TLR1 30 10 8 27 0 7 6 20 0 6 100TLR10 44 6 4 9 0 4 3 7 0 3 100TLR2 9 8 5 56 0 4 2 22 0 2 100TLR3 11 7 5 45 0 2 1 9 0 1 100TLR4 14 6 5 36 0 4 3 21 0 3 100TLR5 54 13 7 13 0 10 5 9 2 5 100TLR7 59 14 5 8 0 13 5 8 0 5 100TLR8 43 14 7 16 0 11 6 14 0 5 83TNF 6 8 5 83 0 4 2 33 0 2 100TNFAIP1 11 7 5 45 0 4 3 27 0 3 100TNFRSF1A 29 12 6 21 4 11 6 21 3 6 100TOLLIP 48 8 4 8 0 8 4 8 0 4 100TRAF6 34 12 7 21 0 10 6 18 0 6 100TRPV5 88 8 5 6 0 7 4 5 0 4 100VCAM1 40 7 4 10 0 7 4 10 0 4 100VEGF 33 12 6 18 0 12 6 18 0 6 100VTN 12 6 5 42 0 2 1 8 0 1 100Totals 3,750 883 538 14 118 658 379 10 39 372 95

The first column lists the gene name and the second column reports the total number of SNPs in each gene. The following two blocks of four columns reportthe number of haplotypes (Haplotypes), the number of htSNPs (htSNPs), the proportion of htSNPs with respect to the total number of SNPs in the gene (Ratio),and the execution time in seconds (Time), for the African-American sample and the European-American sample. The last two columns report the absolute number(Number) and the proportion (Ratio) of htSNPs in the European-American sample also found in the African-American sample. For example, the first line reportsthat the haplotype set of gene ACE2 contains 57 SNPs, 13 haplotypes were identified in the African-American sample, 7 SNPs are sufficient to identify thesehaplotypes (12% of the original 57 SNPs), and it took �1 sec to identify them. The last two columns report that 6 tagging SNPs are shared between theAfrican-American and the European-American samples, 100% of the tagging SNPs of the European-American sample.

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