From ATP to Electron Transfer: Electrostatics and Free-Energy

© Copyright 2001 by the American Chemical Society

VOLUME 105, NUMBER 23, JUNE 14, 2001

FEATURE ARTICLE From ATP to Electron Transfer: Electrostatics and Free-Energy Transduction in Nitrogenase I. V. Kurnikov, A. K. Charnley, and D. N. Beratan* Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260 ReceiVed: July 18, 2000; In Final Form: February 14, 2001

Nitrogenase consists of two proteins that work in concert to reduce atmospheric dinitrogen to a biologically useful form, ammonia (Curr. Opin. Chem. Biol. 2000, 4, 559-566; Chem. ReV. 1996, 96, 2965-2982). The smaller of the proteins (the so-called Fe protein) shuttles high-energy electrons to the larger subunit (the so-called MoFe protein) where the reduction of dinitrogen molecules takes place. The Fe protein catalyzes the hydrolysis of two MgATP molecules per electron transferred to the MoFe protein. The physical mechanism that couples the ATP hydrolysis and electron-transfer reactions in nitrogenase is one of the “great mysteries” of nitrogen fixation. Our goal is to describe the free-energy transformations that occur in nitrogenase based upon theoretical analysis of structural and electrochemical data. The electrostatic and thermodynamic analysis described here, made possible by recent X-ray structural data (and motivated by closely related electrochemical studies: Biochemistry 1997, 36, 12976-12983; FEBS Lett. 1998, 432, 55-58), shows that the ATP hydrolysis energy in nitrogenase serves the purpose of increasing the driving force of the electron-transfer reaction in the protein-protein complex. MgATP binding induces conformational changes and protein-protein association. The protein-protein docking excludes water from the negatively charged [Fe4S4]Scys 4 redox cofactor that lies near the Fe-protein surface, boosting its energy through diminished solvation. We estimated the induced redox-potential change to be equal to or larger than one-third of an electronvolt, which is roughly the energy associated with the hydrolysis of one MgATP molecule. Nitrogenase appears, therefore, to employ a relatively simple ATP hydrolysis coupled redox cofactor desolvation mechanism to energize, and thus to accelerate, interprotein electron transfer. Our analysis also indicates that electrostatic interactions play an important role in the substitution of MgADP by MgATP upon reduction of the [Fe4S4]Scys 4 cluster in the Fe protein. The nitrogenase scheme of energy conversion may suggest alternative strategies for the design of new molecular devices.

I. Biological Free Energy: Electrochemical Potential and ATP In biology, the free energy that is used to power chemical reactions is stored in many forms. These include transmembrane electrochemical gradients, chemical species with labile bonds, and redox-active species.5 The mobile electrons, shuttled between the redox-active species, reside on specific chemical

groups known as redox cofactors. Biological redox cofactors function in a relatively narrow range of electronic energies. Midpoint potentials of these cofactors (iron-sulfur clusters, hemes, quinones, etc.) occur in a window of about 1 V centered around 0 V vs the normal-hydrogen electrode. Special redox species, including aromatic amino acid side chains and the terminal redox species in photosynthesis, widen this range to 0 ( 1 V.5 The individual reduction-oxidation (redox) potentials,

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E°, correspond to the donor (D) and acceptor (A) half reactions D - e- h D+ and A+ + e- h A. A large positive redox potential corresponds to a “strong” (or highly oxidizing) acceptor, while a large negative potential corresponds to a highly reducing donor. Thus, a thermodynamically “downhill” or spontaneous process occurs when ∆E° is positive, where ∆E° ) [E°(A+ + e- h A) - E°(D - e- h D+)]. The free energy of a redox reaction is described by the Nernst equation:

∆G° ) -ne∆E°

(1)

where n is the number of electrons transferred (equal to 1 in the cases described here), e is the magnitude of the electron charge, and ∆E° is the standard redox potential for products minus reactants, measured in volts. Most energies reported here are in electronvolt units (23.06 kcal/mol ) 1 eV). Simple electron-transfer proteins shuttle electrons to generate transmembrane electrochemical potential gradients as well as to specific enzymes that carry out chemical oxidation and reduction reactions. Typical biological electron-transfer steps occur over 5-15 Å. At these distances, donor and acceptor groups are beyond direct (van der Waals) contact so the electrontransfer (ET) rate is described in a nonadiabatic framework as

kET )

2π 1 |H |2 exp[-(∆G° + λ)2/4λkBT] (2) p DA 4πλk T x B

The rates depend exponentially on two factors: the length of the dominant coupling pathway linking donor to acceptor, reflected in the coupling element HDA,6 and the activation free energy, (∆G° + λ)2/4λ, where ∆G° is the reaction free energy and λ is the reaction reorganization energy.7 For each chemical bond introduced into a donor-acceptor coupling pathway, the ET rate drops by about a factor of 3. For each increase in driving force (-∆G°) of 100 mV, the rate increases by an order of magnitude (for typical λ values of 1 eV and |∆G°| , λ). Thus, for typical spontaneous electron-transfer reactions, the free energy consumed speeds up the rates and can help to ensure unidirectional ET. Another biological form of free-energy storage is the compound adenosine triphosphate (ATP) (usually associated with Mg2+), which can be hydrolyzed with an energy release of approximately one-third of an electronvolt (eV), or ∼7 kcal/ mol. One goal of this paper is to examine how MgATP hydrolysis energy is converted into redox energy (by decreasing the redox potential of the donor species), thus directing the energy of ATP hydrolysis into a specific biosynthetic pathway. Our focus is the MgATP-catalyzed electron-transfer reaction in nitrogenase, a process in which eight single-electron-transfer events occur, each one coupled to the hydrolysis of two MgATP molecules. Proper timing is essential to generate the needed intermediates, at room temperature and atmospheric pressure, along the multi-electron pathway from N2 to NH3. To control N2 fixation, the enzyme has evolved a means of regulating electron delivery; the cost for rapid delivery of highly reducing electrons is apparently ATP hydrolysis. One of the mysteries of nitrogenase function is how the energy of phosphate bond hydrolysis is coupled to the electron-transfer reaction.9 We argue that the key aspect of energy transduction arises from lowering of a (donor) redox cofactor potential upon binding of the two nitrogenase proteins with one another. This increased -∆G° for the ET reaction ensures fast and unidirectional ET between the proteins. We argue further that these large redox potential shifts (as well as associated changes in MgATP and

MgADP binding propensity) can be understood in the framework of fairly simple electrostatic interactions. Section II of this paper outlines the general thermodynamic aspects of energy transduction involving bimolecular proteinprotein electron transfer. Critical to the mechanism are (1) the large charges on the redox cofactors, (2) the location of one cofactor near a protein surface in the free protein, and (3) the fact that the cofactor at the surface in the free protein is buried in the ET active protein-protein complex. Section III describes the continuum electrostatic methods used to examine the changes in energetics during the reaction cycle. Finally, Section IV describes calculations of redox potential shifts upon proteinprotein docking, and the redox-state dependent propensity for MgATP/ADP binding in one of the nitrogenase proteins. We summarize our conclusions in section V, and contrast the energy transduction in nitrogenase to more “direct” strategies, such as those in photosynthesis. The nitrogenase energy transduction mechanism suggests a new strategy for energizing artificial molecular-scale devices. II. Energy and Electron Flow in Nitrogenase The nitrogenase complex consists of two proteins.1,2 The smaller ∼60 kD “Fe protein” contains an [Fe4S4]Scys 4 cluster that can be reduced in vitro by dithionite or by low-potential ET carriers such as ferredoxin or flavodoxin. The Fe protein binds two MgATP molecules, which are hydrolyzed upon delivery of this electron to a second ∼240 kD protein, known as the “MoFe protein”. The MoFe protein contains an ironsulfur P-cluster (with seven iron and eight sulfur atoms), presumably the intermediate electron acceptor, and the catalytic MoFe cofactor (with one molybdenum, seven irons, eight sulfurs, and a homocitrate group).2 The cluster structures and their locations in the nitrogenase complex are indicated in Figure 1. Here, we investigate the two nitrogenase proteins from Azotobacter Vinelandii known as Av2 (the smaller Fe protein) and Av1 (the larger MoFe protein). The overall nitrogen fixing reaction carried out in nitrogenase is

N2 + 8H+ + 8e- + 16MgATP f 2NH3 + H2 + 16MgADP + 16Pi (3) This reaction is exergonic by approximately 15.2 kcal/mol, not including the driving force provided by the hydrolysis of 16 MgATP molecules.8 A substantial free-energy price is presumably paid in this reaction to overcome kinetic barriers to nitrogen reduction at room temperature and atmospheric pressure. The fact that two MgATP molecules are hydrolyzed per electron transferred from Av2 to Av1 indicates that MgATP somehow participates in the molecular recognition and electron-transfer process. Yet, the role of MgATP and the mechanism that couples MgATP hydrolysis to the ET reaction “remains one of the great mysteries of the mechanism of nitrogen fixation”,9 although dramatic progress in this field has occurred in the last 5 years.1 Figure 2 summarizes a working model for a cycle of MgATP hydrolysis-coupled electron transfer between the Fe protein (Av2) and the MoFe protein (Av1) of nitrogenase, which can be drawn from experimental data. (It should be noted that some of the details of this scheme remain the subject of debate. Nonetheless, this outline provides focus for the mechanistic discussions that follow.) The nitrogenase cycle starts with Av2 and Av1 in the uncomplexed state, the [Fe4S4]Scys 4 cluster of Av2 in the oxidized state, and two MgADP molecules bound to Av2, blocking binding of MgATP. In the first step of the reaction, the [Fe4S4]Scys 4 cluster of Av2 is reduced by a strong

Feature Article

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Figure 1. (a) Structures of the [Fe4S4]Scys 4 cluster (Av2), the P-cluster (Av1), and the MoFe cofactor (Av1) (after refs 22 and 41). Depending on its charge state, the P-cluster can exist in two forms: one not bonded to Ser β188 and the other coordinated by the oxygen of deprotonated Ser β188. (b) X-ray structure of the Av1-Av2 complex,11 indicating the relative positions of the complexes, is shown.

Figure 2. Overall reaction scheme for nitrogenase: (1) reduction of Av2 by ferredoxin, (2) ATP/ADP exchange in Av2, (3) Av2-Av1 complex formation, (4) electron transfer from Av2 to Av1, (4) ATP hydrolysis, and (5) complex separation.

reducing agent (such as sodium dithionite or a low-potential electron-transfer protein). The formal charge of the [Fe4S4]Scys 4 cluster changes on reduction from (-2e) to (-3e). The binding cluster of MgADP to Av2 is destabilized upon [Fe4S4]Scys 4 reduction. In the second step, MgADP is substituted by MgATP, which causes Av2 to adopt a more compact conformation, as

indicated by a reduced radius of gyration observed in low-angle X-ray scattering experiments.10 Av2 in this compact conformation (with two MgATP molecules attached) binds to Av1 (step 3). In the Av2-Av1 complex, the [Fe4S4]Scys 4 cluster of Av2 is in van der Waals contact with Av1 at the surface position nearest the P-cluster of Av1 (approximately 14.5 Å between nearest

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Kurnikov et al. (D) (A) (D) ∆∆Grxn ) -e(E(A) cplx - Ecplx) + e(Esoln - Esoln) (4) rxn - G(3) - G(2) - G(1)) ∆∆Grxn ) ∆Grxn cplx - ∆Gsoln ) (G bind bind - ∆GD-A ) ∆∆Gbind ) ∆GDA-

(G(4) - G(2) - G(3) - G(1)) ) ∆∆Grxn (4)

Figure 3. Thermodynamic cycle associated with Av1-Av2 binding and ET. Equation 2 shows that the redox potential change upon protein-protein binding is equal to the increase in complex binding energy following electron transfer.

cluster metal atoms, based on X-ray data11), optimizing conditions for electronic coupling between these clusters. An electrontransfer reaction between the [Fe4S4]Scys 4 cluster of Av2 and the P-cluster of Av1 occurs in the complex (step 4). While there is some debate as to precise timing, the hydrolysis of two ATP molecules bound to Av2 occurs (step 5) and inorganic phosphate is released to solution. Phosphate release destabilizes the Av2Av1 complex, leading to dissociation (step 6) that restores the initial state of the system. Critical experimental observations3,4 established that the redox potential of the Av2 [Fe4S4]Scys 4 cluster is lowered by several hundred millivolts, relative to the redox potential of the P-cluster, upon Av2-Av1 complex formation. Thus, ET from Av2 to Av1 obtains a free-energy boost that is irreversibly consumed in the course of the electron-transfer reaction. As we show in detail below, the spent free energy must be “repaid” to dissociate the complex. This free energy is derived, finally, from the hydrolysis of ATP, which is the only replenishable source of free energy in the system. However, in the course of the reaction, several transformations of free energy take place. Our main goal is to build an understanding of these transformations based upon theoretical analysis. An ATP-free deletion mutant L127∆ of Av2 (which is believed to adopt a compact conformation resembling that of Av2 with MgATP bound) also forms a complex with Av1.12 In this special complex, a single-electron transfer from reduced Av2 to Av1 can occur without ATP hydrolysis. However, the Av2-Av1 complex of L127∆ does not dissociate following ET. Tightening of the Av1-Av2 complex after ET is a necessary consequence of boosting the driving force of the ET reaction between Av2 and Av1 on complex formation and of energy conservation. This can be proven by considering the thermodynamic cycle in Figure 3, involving the four states: (1) D- + A, (2) D + A-, (3) [D-|A], and (4) [D|A-] (where [‚|‚] represents a protein-protein complex). Denoting the free energy of the system in state (i) as G(i) we find the change in the ET reaction free energy upon protein-protein binding (∆∆Grxn) and the change in the binding energy of the protein-protein complex upon ET (∆∆Gbind):

(A) (D) (A) Here E(D) cplx, Ecplx, Esoln, and Esoln are redox potentials of the donor and acceptor cofactors in the complex and solution, respectively. The increase in reaction free energies is linked to the change in redox potentials through the Nernst equation. The change in ET driving force upon binding is therefore equal to the change in complex binding energy after ET. Four-state thermodynamic cycles of this kind are used widely in bioenergetics.13 The increase in the electron-transfer reaction free energy upon protein-protein binding (∆∆Grxn)leads directly to an equal increase in the binding energy of the protein-protein complex (∆∆Gbind) after ET from Av2 to Av1. If MgATP were not allowed to hydrolyze, the Av2-Av1 complex would not dissociate (as demonstrated as well in experiments with nonhydrolyzable ATP analogues3). Thus, an increase in the driving force of the Av2-Av1 ET reaction seems to occur at the expense of increasing the Av2-Av1 (and MgATP-Av2) binding free energy. ATP hydrolysis leads to breaking apart the tightened Av2-Av1 complex, because the release of phosphate shifts the equilibrium of the system toward the dissociated state (step 6 of Figure 2). As the overall scheme of MgATP hydrolysis-coupled ET from Av2 to Av1 becomes clearer, the focus of studies must move to the elucidation of the detailed molecular interactions controlling the transformations depicted in Figure 2. As discussed above, changes to the [Fe4S4]Scys 4 cluster redox potential upon Av2-Av1 complex formation and MgATP or MgADP binding are critical for energy transduction. Redox potentials of cofactors in proteins are affected greatly by electrostatic interactions among the charges of the cofactors, the surrounding protein, and the solvent. It is challenging to identify contributions to redox potentials directly from experiment, although recent studies14 are aimed at isolating contributions to the redox cluster using mutagenesis. We potential of the [Fe4S4]Scys 4 describe below theoretical investigations of how electrostatic interactions affect redox potentials of the Av2 and Av1 cofactors. We also provide quantitative justification, based on continuum-dielectric models and numerical solutions of the Poisson equation to estimate electrostatic interaction energies, for the qualitative arguments just made. The specific questions that we address in nitrogenase are: What interactions cause changes to the redox potential of the [Fe4S4]Scys 4 cluster, boosting the driving force for interprotein electron transfer? What triggers initial ADP/ATP binding exchange in the iron protein? What is the mechanism for dissociation of the Av2-Av1 complex? The analysis described below indicates that electrostatic interactions play a key role in the physical mechanism that couples MgATP hydrolysis to ET in the nitrogenase complex.

III. Theoretical Methods Our analysis employs finite-difference solutions of the Poisson equation (eq 5) to compute electric fields and electrostatic energies associated with the nitrogenase proteins and their complexes.

∇‚[(r)∇φ(r)] ) - 4πF(r)

(5)

Feature Article In this analysis, the protein is treated as a continuous structureless low-dielectric medium with imbedded charges surrounded by a high-dielectric aqueous region. Charge distributions on the amino acids are represented by a set of atomic charges. Calculations of this kind and more microscopic models have proven to be extremely useful to interpret protein redox potentials, pKa’s, and reaction mechanisms.15-18 The computations described below were performed using the program DelPhi.19 The Poisson equation is solved on a rectangular 255 × 255 × 255 lattice, where the spacing between lattice points in each dimension is determined by the protein size and shape. Along each box dimension, the protein covers 60% of the lattice points. In these computations, the density of grid points was about two per ångstrom. To test the convergence of the calculated results with respect to the grid spacing and the number of grid points, we also performed calculations with a smaller number of grid points (121 × 121 × 121 lattice) and a larger overall box size (with each linear dimension of the box 3 times larger than the largest dimension of the protein). Coulomb boundary conditions were employed in the calculations. [The coulomb boundary conditions set the potential at the boundary points of the grid according to Coulomb’s law for the protein charges in a uniform medium having a dielectric constant of 80.] We set atomic charge parameters for the amino acids using the AMBER force-field database.20 Asp, Glu, Lys, and Arg residues were assumed to be charged; histidines were assumed unprotonated. We determined atomic charge parameters for ATP, ADP, HPO4-2, and the P-cluster of Av1 using HartreeFock calculations (3-21G basis set and Hay-Wadt pseudopotential for the Fe atoms) and the Merz-Kollman charge-fitting scheme.21 To model the charges of the Av2 [Fe4S4]Scys 4 cluster, we distributed a -2 or -3 charge uniformly on the 12 atoms of the cluster. The P-cluster can exist in at least two structural forms that differ in coordination by the Ser β 188 residue and the protonation state of this residue. In our calculations, we used the form of the P-cluster that lacks coordination by the Ser β 188 Oγ atom (see Figure 1a), and this Ser was assumed to be in its canonical form. This structure corresponds to the so-called PN redox state.22 The nature of the P-cluster electron accepting state(s) remains the subject of ongoing investigation. In the calculation of the redox-potential change, the redox states of the P-cluster were assumed to be PN (formal charge of -4) and P1+ (formal charge of -3). At the level of this simple electrostatic analysis, details of the cluster charge distribution do not influence our basic conclusions. Results obtained in the electrostatic analysis using quantum chemistry derived P-cluster charge parameters were not substantially different from those determined with a simple uniform P-cluster charge distribution. Because the P-cluster is buried below the Av1 surface by more than 10 Å, its redox potential shifts upon complex formation are small compared to the shifts in the (initially surface exposed, buried in the complex) Av2 cluster. Since we are mainly interested in redox potential shifts of the Av2 cofactor upon docking, the details chosen for the P-cluster structure are of limited importance. The separation surface between protein and solvent (regions of low- and high-dielectric constants) were calculated using the Connolly algorithm.23 The boundary depends on the choice of atomic radii parameters; PARSE atomic radii13 optimized to reproduce hydration solvation energies for small organic compound were used in this study. The radii of iron atoms (not present in the PARSE set) were set to 2.0 Å. In addition to atomic charges and radii, the other major input

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Figure 4. Thermodynamic cycle used to calculate the redox potential shifts for the iron-sulfur cluster upon transfer from a continuum solvent to a protein dielectric environment.

parameter for the electrostatic computations is the protein dielectric constant, p. The issue of the most appropriate value for the protein dielectric parameter remains the subject of considerable debate.24-27 The polarizability of the protein and, correspondingly, the local dielectric constant p varies among different proteins and even within the same protein. The effective dielectric constant may be larger at the protein surface than in the core.24,27 We have performed calculations with p equal to 4 and 10, representing lower and upper bounds of protein dielectric constants found in the literature.24,27 The surrounding solution dielectric constant was fixed equal to 80 (water). Despite the uncertainty in the value of p, robust qualitative conclusions concerning the importance of electrostatic interactions associated with energy transduction in nitrogenase can be drawn from this analysis. We first calculate the shift in the redox potential of the [Fe4S4]Scys 4 cluster of Av2 and the P-cluster of Av1 upon transfer of these species from an aqueous solvent environment into their respective protein environments, using the four-state thermodynamic cycle depicted in Figure 4. For each of the systems, and charge distributions depicted in Figure 4 (two states are the protein surrounded by water with oxidized or reduced clusters; the other two states are reduced or oxidized clusters in aqueous solvent without protein), the total electrostatic energies were calculated using potentials computed from solutions of the Poisson equation on the mesh and eq 628

Etot el )

1 2

∑i qiφi

(6)

The sum in eq 6 is performed over all grid points, qi is the charge at each point i, and φi is the electrostatic potential calculated at the grid point. Electrostatic energies are directly related to redox potential shifts through

-e∆E°waterfprotein) (Ered-prot - Eox-prot )el el - Eox-water ) (7) (Ered-water el el The protein structures used in this modeling study were drawn from available X-ray data. Protein databank (PDB) entry 2NIP29 was used to model the nucleotide-free Av2 protein. The structure of Av2 with two MgADP’s bound was modeled with the PDB structure 1FP6;30 the Av1-Av2 complex with two MgATP’s bound was modeled from the PDB structure 1N2C,11 which has

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a TABLE 1: Electrostatic Contributions (Eq 7) to the Redox Potential (V) of the [Fe4S4]Scys 4 Cluster in Av2

contribs to ∆E ° of [Fe4S4]Scys 4 ∆E Mg ° 2+ due to ∆E MgANP ° dir due to MgANP ∆E °prot due to protein charges ∆E °solv desolvation contribs (eq 7)

Av2 (nuc free)

Av2 (2MgADP)

Av2 (2MgATP)

Av1-Av2 cplx (2MgATP)

+0.38 (+0.16) (a) -0.78 (-0.32) (b)

+0.07 (+0.06) -0.02 (-0.02) (c) +0.38 (+0.14) (d) -0.83 (-0.34) (e)

+0.11 (+0.07) -0.08 (-0.05) (f) +0.34 (+0.16) (g) -0.70 (-0.30) (h)

+0.24 (+0.13) -0.13 (-0.07) (i) +0.38 (+0.09) (j) -1.66 (-0.62) (k)

Mg2+

a Specific contributions arising from protein charged groups and from bound species are indicated. Calculations were performed for protein dielectric constants of 4 and 10 (in parentheses). Letters in parentheses are used in reference to computations in Table 2.

TABLE 2: Calculated Shifts (V) in Redox Potential of the [Fe4S4]Scys 4 Cluster upon Binding of MgADP or MgATP to Av2 and Complex Formationa ∆E MgADP ° due to 2MgADP binding [c + (d - a) + (e - b)] ∆E °MgATP due to 2MgATP binding [f + (g - a) + (h - b)] ∆E °complex form due to the formation of the Av2-Av1 complex [(i - f) + (j - g) + (k - h)] a

calcd

exptl

-0.07 (-0.06) -0.04 (-0.03) -0.96 (-0.41)

-0.1237,38 -0.1037,38