Chapter 13

Metalloproteins to Membrane Proteins: Biological Energy Transduction Mechanisms

Douglas C. Rees

Division of Chemistry and Chemical Engineering 147-75CH, Howard Hughes Medical Institute, California Institute of Technology,

Pasadena, CA 91125

Structural analyses of two macromolecular systems, the metalloprotein nitrogenase and the integral membrane protein MscL (mechanosensitive channel of large conductance), are discussed within the context of energy transduction mechanisms. Nitrogenase catalyzes the ATP dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation, while MscL is a stretch activated (mechanosensitive) channel that opens and closes in response to changes in lateral tension applied to membranes. Although nitrogenase and MscL have very different structures and functions, they both mediate the coupling of two energetic processes. From these studies, it is suggested that effective coupling of two processes by transduction proteins occurs through conformational states common to each process.

It is a special pleasure to have the opportunity to participate in this symposium in honor of William N. Lipscomb's 80th birthday. The breadth of the Colonel's interests and the significance of his contributions remain a continual source of amazement and inspiration to those of us fortunate to have

202 © 2002 American Chemical Society

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been in his group. Perhaps even more remarkably, the Colonel's talents are combined with a remarkable sense of humor. One example of many provided at the symposium were the research propositions submitted by the Colonel in fulfillment of the requirements for the PhD in Chemistry at Caltech (i). In particular, the last prop notes:

"1 l.(a) Research and study at the Institute have been unnecessarily hampered by the present policy of not heating the buildings on weekends.

(b) Manure should not be used as a fertilizer on ground adjacent to the Campus Coffee Shop."

Another aspect of the Colonel's thesis that attracted considerable attention from my group was that about half of his thesis work, as well as two more props, were war-related and hence classified so that they do not appear with the deposited copy of his thesis - which immediately suggested a way to cut the writing time for the thesis or props in half.

My interest in protein structure originated while I was a graduate student in the Colonel's group. My first exposures to both macromolecular crystallography and metalloproteins were provided by my thesis research on the structural analysis of the binding of inhibitors, both small molecule and protein, to the metalloprotein carboxypeptidase A. The structure of carboxypeptidase A, established in the Colonel's group, was the first to be determined for a metalloenzyme, and this system provided a fascinating opportunity to combine structure, mechanism and metals. My future research interests in nitrogenase also grew out of my experiences in the Colonel's lab through interactions with Jim Howard of the University of Minnesota. Jim was spending his sabbatical at Harvard and through numerous discussions, he convinced me that nitrogenase was of sufficient interest that I subsequently postdoc'ed with him, and we have continued our collaboration in this area ever since.

Metalloproteins and membrane proteins represent extremely broad classifications of proteins with diverse biological roles; this article cannot begin to adequately introduce either area, much less both. Instead, one example each of a metalloprotein (nitrogenase) and a membrane protein (MscL) will be described, emphasizing the functional implications of the structural studies, particularly in the context of energy transduction mechanisms.

Energy transduction processes

Life depends on energy transduction processes that couple cellular metabolism to environmental energy sources such as light or reduced compounds. These primary energy sources must be efficiently converted into

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biologically usable forms such as concentration gradients or ATP, to fuel the molecular processes required for the growth and survival of organisms, including biosynthesis, transport, motility, etc. The common elements in an energy transduction process involve the coupling of an energetically favorable process to drive an energetically unfavorable process. In the case of bacteriorhodopsin, for example, the energetically favorable absorption of light is used to drive the formation of a pH gradient across the cellular membrane. Proteins mediating these interconversions represent attractive targets for structural studies since they exist in multiple conformations and often have been extensively studied by a variety of biochemical and biophysical approaches. As a result, there has been an explosion of activity in the structural characterization of transduction proteins in recent years (see (2)). Analysis of these systems indicates that a key feature of energy transduction mechanisms is that the two processes must be coupled; this can be achieved by having the biological system exclusively undergo a common, coupled transition while suppressing the independent reactions. In this fashion, "slippage" or "short-circuiting" of the transduction process will be minimized. The role of the protein in the transduction process is to kinetically facilitate the coupled reaction, while simultaneously making the uncoupled (independent) reactions kinetically unfavorable. The structural basis underlying these coupling mechanisms will be analyzed in more detail in the following sections for two specific systems: the metalloprotein nitrogenase and the membrane protein channel MscL.

Metalloproteins: Nitrogenase

Although the earth is surrounded by an atmosphere composed predominantly of dinitrogen, most organisms are unable to utilize this source for metabolic purposes. Fortunately, there are a specialized group of organisms known as nitrogen fixers or diazotrophs that are able to catalytically reduce dinitrogen to the metabolically usable form of ammonia. Organisms responsible for biological nitrogen fixation contain the nitrogenase enzyme system (reviewed in (3-8)) that consists of two component metalloproteins, the iron (Fe-) protein and the molybdenum iron (MoFe-) protein. Together, these two proteins mediate the ATP dependent reduction of atmospheric dinitrogen to ammonia. Prior to the development of the Haber-Bosch process, biological nitrogen fixation accounted for the majority of fixed nitrogen entering the biosphere (with the remainder provided by lightning and volcanic emissions); nearly a century after the development of the Haber-Bosch process, industrial ammonia formation represents one of the largest chemical industries and produces comparable amounts of ammonia to the biological process. Indeed, the introduction of the Haber-Bosch process has had a revolutionary impact on the ability of the earth to sustain substantial population increases over the past century (9).

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The basic outlines of the electron flux through the nitrogenase system have been established, and involve the initial reduction of Fe-protein by low potential carriers such as ferredoxin or flavodoxin. The Fe-protein then transfers electrons to the MoFe-protein in a process that is coupled to the hydrolysis of ATP. After the appropriate numbers of electrons and protons are present in the MoFe-protein, substrate reduction can take place on the FeMo-cofactor. Despite extensive studies, the overall stoichiometry of the nitrogenase catalyzed reaction has still not been definitively established. The uncertainties are expressed in the following equation for the overall enzyme reaction (8):

N 2 + (6+2n)H+ + (6+2n)e"+ p(6+2n)ATP —> 2NH3 + nit + p(6+2n)ADP + p(6+2n)Pi

In the "standard" model, the evolution of one molecule of dihydrogen is coupled to the reduction of one molecule of dinitrogen, and two molecules of ATP are hydrolyzed per electron transferred, so that n=l and p=2. However, in typical experiments, larger values of both η and ρ are observed. There are also reports of ratios approaching 1 when an all-ferrous form of Fe-protein is used as the electron source (10).

The structures of the component proteins have been determined, both individually and complexed together. The MoFe protein is an α 2 β 2

heterotetramer, where the α and β subunits exhibit similar polypeptide folds that consist of three domains. This protein contains two copies each of two different types of unusual metallocenters (Figure 1), the FeMo-cofactor, the substrate reduction site, and the P-cluster, believed to participate in electron transfer from the Fe protein to the FeMo-cofactor. Each cluster contains 8 metals and associated sulfurs in distinctive arrangements, that have neither been observed in any other enzymes nor modeled synthetically. These metalloclusters are coordinated by ligands contributed by different domains of the MoFe-protein subunits. Intriguingly, the ligands are all present in a common core structure found in each domain that is composed of a four stranded, parallel β-sheet flanked by α-helices (8); a similar structural organization is also observed in the coordination of the Η-cluster at the active site of iron only hydrogenases and in the so-called prismane protein. Although multiple oxidation states have been observed for the clusters, the numbers of electrons associated with a given oxidation state have not been unambiguously assigned. It appears likely, however, that the iron atoms in the as-isolated state of the P-cluster are all ferrous, and are predominantly in the ferrous form in the FeMo-cofactor.

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Figure L Structural models for the nitrogenase metalloclusters and coordinating ligands. (a) the oxidized Pox state of the P-cluster. (b) the

dithionite reduced PN form of the P-cluster. (c) FeMo-cofactor. Protein Data Bank (11, 12) coordinate sets 2MIN and 3MIN were used for (a) and (b)-(c),

respectively. Molecular figures in this chapter were prepared with MOLSCRIPT(13).

The Fe-protein is a dimer of two identical subunits that symmetrically coordinate a single [4Fe:4S] cluster. The isolated Fe-protein can bind MgADP or MgATP at a stoichiometry of two nueleotides/dimer, and participates intimately in the coupling between ATP hydrolysis and electron transfer to the MoFe-protein. Structurally, Fe-protein adopts a polypeptide fold characteristic of P-loop containing nucleotide binding proteins, termed "switch proteins", such as ras, G-proteins and related proteins. These similarities extend to the switch I and II regions, that are conformationally sensitive to the presence or absence of the nucleotide γ-phosphate in the G-protein family. Importantly, one of the cluster ligands, Cys 132, is contained within the switch II region of the Fe-protein and so identifies a possible mechanism for coupling ATP hydrolysis and [4Fe:4S] cluster redox behavior through conformational changes in this region. The Fe-protein cluster exhibits the unusual ability to exist in three oxidation states, including the unprecedented all-ferrous form fl^Fe^S]*0). The structural factors influencing this redox versatility have not been definitively established, but it has been proposed that solvent accessibility of this cluster, unusual for these typically buried clusters, may be a factor (14).

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Mechanistic considerations

At the protein level, the basic mechanism of nitrogenase involves (1) complex formation between the reduced Fe-protein with two bound ATP and the MoFe-protein; (2) electron transfer between the two proteins coupled to the hydrolysis of ATP; (3) dissociation of the Fe-protein accompanied by re-reduction and exchange of ATP for ADP and (4) repetition of this cycle until sufficient numbers of electrons and protons have been accumulated so that available substrates can be reduced. Key mechanistic questions concern the chemical mechanism of the reduction of dinitrogen and other substrates, and the requirement for ATP hydrolysis in this process.

Substrate reduction is generally regarded as occurring at the FeMo-cofactor through a likely sequence of two electron transfers coupled to proton transfers (75). However, little experimental data is available that directly addresses these mechanistic issues, including whether N 2 binds to the FeMo-cofactor primarily at the Mo or one or more Fe sites (indeed, there is no direct experimental evidence to show that N 2 really does bind to the FeMo-cofactor); whether the reduction of any substrate (such as protons) could take place at the P-cluster; how the electron and proton transfers are coupled; and the sequence of intermediates that are generated during substrate reduction. Establishing these mechanistic features remains a priority of nitrogenase research.

Relatively speaking, the role of ATP in the nitrogenase reaction is better characterized than the chemical mechanism of substrate reduction, although many questions remain. Since the reduction of dinitrogen to ammonia is thermodynamically favored at pH 7 when coupled to the oxidation of reduced ferredoxin (16), ATP hydrolysis is not required to provide a thermodynamic driving force for the reaction. Instead, it seems likely that ATP hydrolysis plays a role in the kinetic mechanism of nitrogenase. Key to understanding the role of ATP in the mechanism of nitrogenase is the protein-protein complex between the MoFe-protein and Fe-protein, since it is in this species that ATP hydrolysis is coupled to interprotein electron transfer. We have determined the crystal structures of two stable forms of Fe-protein - MoFe-protein complexes (77, 75), prepared by either running the nitrogenase reaction in the presence of A1F4", resulting in formation of the ADP»A1F4" stabilized complex (19), or through complex formation in the absence of nucleotide with the Leu 127Δ deletion mutant of the Fe-protein (20).

To a first approximation, the interface between the Fe-protein and MoFe-protein are similar in both complexes. The relative positions of the metalloclusters observed in these complex structures indicate that electron transfer from the Fe-protein to the FeMo-cofactor proceeds through the P-clusters (Figure 2); the edge to edge distance of -14Â is compatible with inter­protein electron transfer rates much more rapid (~106 sec"1 (27, 22)) than the

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observed turnover time (-1 sec"1 per N 2 reduced). Indeed, the rate determining step with saturating amounts of substrates has been assigned to dissociation of the protein - protein complex. In both the ADP eAlF 4" and L127A stabilized complexes, the MoFe-protein structures are relatively constant between free and bound forms, while the Fe-proteins have undergone substantial conformational changes. A detailed comparison of the relationships between the various Fe-protein conformations has been presented (18).

Figure 2. A slice through the ADP'AIFS stabilized nitrogenase complex (17) that includes theADP*AlF4~, [4Fe:4S] cluster, P-cluster, and FeMo-cofactor,

illustrating the relationship between the nucleotide binding and electron transfer sites. Relative to the isolated Fe-protein, the change in conformation of the switch II region serves to reposition the Asp 129 sidechains for nucleotide hydrolysis and also moves the [4Fe:4S] cluster closer to the P-cluster. The

linkage of these conformational changes through the switch II region provides the structural basis for the coupling of ATP hydrolysis and electron transfer by

nitrogenase. This figure was prepared from Protein Data Bank set 1N2C.

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Energy Transduction in Nitrogenase

The similarities between Fe-protein and nucleotide switch proteins suggests that one role for nucleotide hydrolysis may be to drive a series of conformational changes that mediate the interactions between Fe-protein and MoFe-protein (see (2, 3)). One of the regions of greatest conformational variability in switch proteins between the NTP and NDP states involves the so-called switch II region. In the case of Fe-protein, the switch II region includes Asp 125, Gly 128, Asp 129 and Cys 132. Consequently, this region contains residues that interact with both the nucleotide and the cluster. Analysis of the nitrogenase complex stabilized by ADP*A1F4" demonstrated that the conformation of switch II that is required for ATP hydrolysis is coupled to a repositioning of the [4Fe:4S] cluster such that the cluster is ~4 Â closer to the MoFe-protein, which should facilitate efficient inter-protein electron transfer by a factor of -100 (2i). As the nucleotides convert between the ATP, transition state, ADP+Pi, ADP, etc. -bound states, the conformation of the switch II region will change, necessarily modulating the electron transfer properties of the nitrogenase proteins and coupling the hydrolysis of ATP to quasi-unidirectional electron transfer from the Fe-protein to the MoFe-protein. These considerations may be critical to ensure the efficient transfer of electrons to dinitrogen by a relatively slow enzyme in the ubiquitous presence of protons that are alternative electron acceptors.

Coupling of ATP hydrolysis to electron transfer in other systems

In addition to nitrogenase, other biochemical systems have been identified that couple ATP hydrolysis to electron transfer. Protochlorophyllide reductase, that catalyzes the stereospecific reduction of the D ring during the light independent pathway for chlorophyll biosynthesis, exhibits striking similarities to the nitrogenase system, particularly in the component analogous to the Fe-protein (23). Furthermore, homologues of the Fe-protein have been identified from the genomic sequences of various microorganisms that are not nitrogen fixers, such as Methanococcus janaschii (24), suggesting that the ability to couple ATP hydrolysis to electron transfer may be of more widespread utility than "just" nitrogen fixation. The activator component (CompA) of 2-hydroxyglutaryl-CoA dehydratase (25) of Acidaminococcus fermentans that participates in glutamate fermentation provides an instructive example of a similar enzymatic activity (ATP driven electron transfer) achieved with a distinct polypeptide fold. As with the Fe-protein, CompA is a dimer of two identical subunits that together coordinate a single [4Fe:4S] cluster (26). In this case, however, the subunits are members of the actin/hexokinase/heat shock 70 family of nucleotide binding proteins, rather than the nucleotide switch protein family, and the nucleotide is bound at the interface between two domains within

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a subunit, rather than at the dimer interface. Despite the differences in folds, in both cases, the cluster is coordinated by both subunits in the dimer; one surface of the cluster is exposed to provide the interaction site with the second component; and the cluster and ATP hydrolysis sites are separated by -20 Â. This arrangement indicates that the nucleotide and redox centers are not directly coupled but rather are indirectly linked through a series of conformational changes driven by ATP hydrolysis and component protein binding to ensure that unidirectional electron transfer takes place at the appropriate time between the redox partners.

Membrane Proteins: MscL

All cells are surrounded by membranes composed of phospholipid bilayers that separate the inside of the cell from the outside world. Embedded within these bilayers are integral membrane proteins that mediate the flow of molecules, information and energy into and out of the cell. One class of these membrane proteins are ion channels that facilitate the transmembrane passage of ions down their electrochemical potential gradient in a process that is central to many signaling and sensing pathways. Functionally important properties of ion channels that we would like to understand in terms of their structures include their conductance (related to the diameter of the permeation pathway), ionic selectivity (most clearly established for the KcsA potassium channel (27)), and the regulation (gating) of channel activity in response to environmental changes by conformational switching between open and closed states.

Mechanosensitive channels open and close in response to mechanical stress applied to the cell membrane or other cytoskeletal component. The best-characterized member is the prokaryotic large-conductance mechanosensitive channel (MscL) that was originally identified, isolated, and characterized in Escherichia coli, primarily through the efforts of Kung and co-workers (28, 29). Our interest in MscL grew out of desire to study a gated channel system to help establish the structural basis for the switching between open and closed states in response to environmental changes. For this purpose, we wanted to study a gated system that was small and prokaryotic, to hopefully simplify expression. These constraints were beautifully satisfied by MscL, perhaps uniquely so at the time this project was initiated.

MscL is a nonselective ion channel of -2.5 nS conductance activated in vitro by the application of membrane tension; with a pqpntial difference of 100 mV, this conductance is equivalent to the flow of -10 ions/second across the membrane. The functional properties of MscL suggest a physiological role in the regulation of osmotic pressure in the cell. Sequence analysis and biochemical studies indicated that the MscL channel consists of a single type of subunit of -16 kD with two transmembrane helices, and that this channel is localized

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within the inner cell membrane with both N H 2 - and C O O H - termini positioned in the cytoplasm. MscL homologs have been identified in other bacteria (30).

We took advantage of the availability of bacterial homologues of MscL to test nine different homologues for expression, purification and crystallization, with the final result that crystals of the MscL homologue from Mycobacterium tuberculosis were obtained that diffracted to 3 .5Â resolution. The structure was subsequently solved by a combination of multiple isomorphous replacement, anomalous scattering and noncrystallographic symmetric averaging (31).

Figure 3 The Tb-MscL pentamer viewed from directions perpendicular (left) and parallel (right) to the normal of the membrane plane, respectively. The vertical bar on the left is 30Â in length and marfa the approximate membrane-spanning

region of these channels.

MscL is organized as a pentamer (Figure 3) composed of two domains, transmembrane and cytoplasmic, that share the same fivefold axis relating subunits within the channel. The transmembrane helices are all tilted - 3 0 ° relative to the membrane normal The first transmembrane helix of each MscL subunit starts in the cytoplasm and crosses the membrane as the "inner" helix to form the permeation pathway of this channel, while the second helix returns to the cytoplasm across the membrane to form the "outer" helix. Hence, MscL is threaded across the membrane in the opposite manner to KcsA. The inner helices pack against each other to form a right-handed helical bundle with a crossing angle of —40°. Each inner helix contacts four adjacent helices (two inner and two outer), while the outer helices contact their own and an adjacent inner helix. The extracellular loop connecting the transmembrane helices exhibits extensive sequence variability within the M s c L family. The cytoplasmic domain consists of a five helix bundle that extends for - 3 5 Â away from the likely plane of the membrane-aqueous interface.

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Figure 4. A helical wheel for the MscL inner helix that illustrates the positions of helix interface residues, pore residues and gain offunction mutations. The sidechain of Vol 21 serves to constrict the channel at the narrowest position.

The correspondence between residues at the interface between TM1 helices and the gain offunction mutations is evident, suggesting this interface is critical for the gating process. TM1 and TM2 are the inner and outer helices, respectively.

An important motivation for initiating the structural analysis of MscL was to understand channel gating, since MscL opens and closes in response to mechanical stresses applied directly to the membrane. The high conductance and lack of ion selectivity are consistent with a large, water-filled pore existing in the open state of the MscL channel. An important development in establishing the gating mechanism of MscL has been the identification of 'gain-of-function' mutants that display a slow or no-growth phenotype as a result of the leakage of solutes out of the cell under low osmotic strength growth conditions (32). Many of the mutations associated with severe phenotypes are located at the interface between adjacent inner helices (Figure 4), in the region of the membrane spanning domain where the pore is most restricted (at the side chain of Val 21 in the closed state). These observations suggest that contacts between inner helices play a crucial role in the gating mechanism. This interface must be rearranged, perhaps by allowing the inner and outer helices to interleave like barrel staves, to create a pore of sufficiently large diameter in the open state. More recently, a detailed model for the closed to open transition of MscL has been proposed by Sukharev and Guy based on a combination of modeling, electrophysiological and disulfide crosslinking experiments (33). This model invokes an extended rearrangement of the helices with the inner and outer helices becoming more tilted and immersed in the bilayer, while a five helix bundle formed by the N-terminal residues of MscL (not observed in the crystal structure) form the true gate that must be opened for conduction. The

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availability of such models will be invaluable for the design of experiments to more conclusively establish the structural organization of this state.

An interesting similarity in the channels studied to date is that the helices lining the permeation pathway pack together as right handed helical bundles with crossing angles (-40°) that are relatively steep for membrane proteins (34). One consequence of this relatively steep packing angle is that the contact interface between helices is localized to a fairly narrow region, which may facilitate helix-helix rearrangements associated with channel gating (35). As with other membrane proteins, residues buried at the interface between interacting transmembrane spanning helices are predominantly apolar, with relatively few hydrogen bonds or other polar types of contacts observed. The apolar and non-directional nature of these helix-helix contacts may also facilitate helix rearrangements associated with channel gating.

A key aspect of the function of MscL is the coupling mechanism between protein conformation and membrane stretching, which is at the heart of the energy transduction. Thermodynamically, if a protein exists in multiple states with varying cross-sectional areas, stretching the membrane will increasingly favor states with the larger cross-sectional areas. In the case of MscL, an applied tension of -12 dynes/cm is required to open the channel, which approaches the tension needed to rupture the membrane. This suggests as a working model that the lateral pressure in the membrane bilayer clamps the channel in the closed state; when the membrane is stretched, this pressure is reduced, allowing the channel to expand to the open state as lipids are no longer as tightly packed against the channel. For a mechanism such as that proposed by Guy and Sukharev, stretching the membrane could lead to bilayer thinning, with the an associated increase in helical tilt to keep the hydrophobic residues immersed in the non-polar region of the membrane bilayer.

Concluding Remarks

Consideration of the general properties of energy transduction mechanisms indicates that there are common elements to their fonction. In particular, the efficient coupling of two processes by energy transduction proteins involves common conformations that are required for both processes. Since these conformations can apparently only be achieved when the complete set of substrates, ligands, etc. are bound to the transduction protein, the coupling efficiency is increased by suppression of uncoupled processes. In real systems, the coupling mechanisms are as diverse as the processes themselves. In the cases discussed in this chapter, nitrogenase is proposed to represent an example of kinetic coupling of ATP hydrolysis and electron transfer to ensure unidirectional electron transfer from the Fe-protein to the MoFe-protein, while MscL likely

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represents a case of thermodynamic coupling where membrane stretching favors the state of largest cross-sectional area (i.e., the open state).

Finally, as an update on the status the Colonel's last research proposition (i), I am pleased to note that while Caltech may not be able to provide air-conditioning this summer, the buildings are heated on weekends in the winter. As far as the use of manure is concerned, I have detected it on rare occasions, but clearly there are still more effective mechanisms for supplying fixed nitrogen to the grounds.

Acknowledgments

I would like to thank the Colonel for his advice and guidance over the years, as well as allowing us to collect the first data sets of the nitrogenase Fe-protein in his lab. None of this work would have been possible without my interactions with Jim Howard, or without the efforts of my group on the projects described from my lab. Research work on nitrogenase and MscL has been supported by NIH grants GM45162 and GM62532, respectively.

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