Electron Transfer In Nitrogenase Research Articles

Electron Transfer in Nitrogenase.

Nitrogenase is the only enzyme capable of reducing N2 to NH3. This challenging reaction requires the coordinated transfer of multiple electrons from the reductase, Fe-protein, to the catalytic component, MoFe-protein, in an ATP-dependent fashion. In the last two decades, there have been significant advances in our understanding of how nitrogenase orchestrates electron transfer (ET) from the Fe-protein to the catalytic site of MoFe-protein and how energy from ATP hydrolysis transduces the ET processes. In this review, we summarize these advances, with focus on the structural and thermodynamic redox properties of nitrogenase component proteins and their complexes, as well as on new insights regarding the mechanism of ET reactions during catalysis and how they are coupled to ATP hydrolysis. We also discuss recently developed chemical, photochemical, and electrochemical methods for uncoupling substrate reduction from ATP hydrolysis, which may provide new avenues for studying the catalytic mechanism of nitrogenase.

Redox-Dependent Metastability of the Nitrogenase P-Cluster.

Molybdenum nitrogenase catalyzes the reduction of dinitrogen into ammonia, which requires the coordinated transfer of eight electrons to the active site cofactor (FeMoco) through the intermediacy of an [8Fe-7S] cluster (P-cluster), both housed in the molybdenum-iron protein (MoFeP). Previous studies on MoFeP from two different organisms, Azotobacter vinelandii ( Av) and Gluconacetobacter diazotrophicus ( Gd), have established that the P-cluster is conformationally flexible and can undergo substantial structural changes upon two-electron oxidation to the POX state, whereby a backbone amidate and an oxygenic residue (Ser or Tyr) ligate to two of the cluster's Fe centers. This redox-dependent change in coordination has been implicated in the conformationally gated electron transfer in nitrogenase. Here, we have investigated the role of the oxygenic ligand in Av MoFeP, which natively contains a Ser ligand (βSer188) to the P-cluster. Three variants were generated in which (1) the oxygenic ligand was eliminated (βSer188Ala), (2) the P-cluster environment was converted to the one in Gd MoFeP (βPhe99Tyr/βSer188Ala), and (3) two oxygenic ligands were simultaneously included (βPhe99Tyr). Our studies have revealed that the P-cluster can become compositionally labile upon oxidation and reversibly lose one or two Fe centers in the absence of the oxygenic ligand, while still retaining wild-type-like dinitrogen reduction activity. Our findings also suggest that Av and Gd MoFePs evolved with specific preferences for Ser and Tyr ligands, respectively, and that the structural control of these ligands must extend beyond the primary and secondary coordination spheres of the P-cluster. The P-cluster adds to the increasing number of examples of inherently labile Fe-S clusters whose compositional instability may be an obligatory feature to enable redox-linked conformational changes to facilitate multielectron redox reactions.

Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From Gluconacetobacter diazotrophicus.

Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensive crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail. In this chapter, we summarize some of our efforts toward obtaining such an understanding. Experimental investigations of the structure-function relationships in nitrogenase are challenged by the fact that it cannot be readily expressed heterologously in nondiazotrophic bacteria, and the purification protocols for nitrogenase are only known for a small number of diazotrophic organisms. Here, we present methods for purifying and characterizing nitrogenase from a new model organism, Gluconacetobacter diazotrophicus. We also describe procedures for observing redox-dependent conformational changes in G. diazotrophicus nitrogenase by X-ray crystallography and electron paramagnetic resonance spectroscopy, which have provided new insights into the redox-dependent conformational gating processes in nitrogenase.

Elucidating thermodynamic parameters for electron transfer proteins using isothermal titration calorimetry: application to the nitrogenase Fe protein.

Establishing thermodynamic parameters for electron transfer reactions involving redox proteins is essential for a complete description of these important reactions. While various methods have been developed for measuring the Gibbs free energy change (Delta G(HR) or E(m)) for the protein half-reactions, deconvolution of the respective contributions of enthalpy (Delta H(HR)) and entropy (Delta S(HR)) changes is much more challenging. In the present work, an approach is developed using isothermal titration calorimetry (ITC) that allows accurate determination of all of these thermodynamic parameters for protein electron transfer half-reactions. The approach was validated for essentially irreversible and reversible electron transfer reactions between well-characterized mediators and between mediators and the protein cytochrome c. In all cases, the measured thermodynamic parameters were in excellent agreement with parameters determined by electrochemical methods. Finally, the calorimetry approach was used to determine thermodynamic parameters for electron transfer reactions of the nitrogenase Fe protein [4Fe-4S](2+/+) couple in the absence or presence of MgADP or MgATP. The E(m) value was found to change from -290 mV in the absence of nucleotides to -381 mV with MgATP and -423 mV with MgADP, consistent with earlier values. For the first time, the enthalpy (Delta H(HR)) and entropy (Delta S(HR)) contributions for each case were established, revealing shifts in the contribution of each thermodynamic parameter induced by nucleotide binding. The results are discussed in the context of current models for electron transfer in nitrogenase.

Electron Transfer in Nitrogenase Analyzed by Marcus Theory: Evidence for Gating by MgATP

Nitrogenase-catalyzed substrate reduction reactions require electron transfer between two component proteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein, in a reaction that is coupled to the hydrolysis of MgATP. In the present work, electron transfer (Marcus) theory has been applied to nitrogenase electron transfer reactions to gain insights into possible roles for MgATP in this reaction. Evidence is presented indicating that an event associated with either MgATP binding or hydrolysis acts to gate electron transfer between the two component proteins. In addition, evidence is presented that the reaction mechanism can be fundamentally changed such that electron transfer becomes rate-limiting by the alteration of a single amino acid within the nitrogenase Fe protein (deletion of Leu 127, L127 Delta). These studies utilized the temperature dependence of intercomponent electron transfer within two different nitrogenase complexes: the wild-type nitrogenase complex that requires MgATP for electron transfer and the L127 Delta Fe protein-MoFe protein complex that does not require MgATP for electron transfer. It was found that the wild-type nitrogenase electron transfer reaction did not conform to Marcus theory, suggesting that an adiabatic event associated with MgATP interaction precedes electron transfer and is rate-limiting. Application of transition state theory provided activation parameters for this rate-limiting step. In contrast, electron transfer from the L127 Delta Fe protein variant to the MoFe protein (which does not require MgATP hydrolysis) was found to be described by Marcus theory, indicating that electron transfer was rate-limiting. Marcus parameters were determined for this reaction with a reorganization energy (lambda) of 2.4 eV, a coupling constant (HAB) of 0.9 cm-1, a free energy change (Delta G' degrees ) of -22.0 kJ/mol, and a donor-acceptor distance (r) of 14 A. These values are consistent with parameters deduced for electron transfer reactions in other protein-protein systems where electron transfer is rate-limiting. Finally, the electron transfer reaction within the L127 Delta Fe protein-MoFe protein complex was found to be reversible. These results are discussed in the context of models for how MgATP interactions might be coupled to electron transfer in nitrogenase.

Changes in the midpoint potentials of the nitrogenase metal centers as a result of iron protein-molybdenum-iron protein complex formation.

All nitrogenase-catalyzed substrate reduction reactions require the transient association between the iron (Fe) protein component and the molybdenum-iron (MoFe) protein component with concomitant intercomponent electron transfer and MgATP hydrolysis. Understanding the effects of Fe protein-MoFe protein complex formation on the properties of the nitrogenase metal centers is thus essential to understanding the electron transfer reactions. This work presents evidence for significant shifts in midpoint potentials for two of the three nitrogenase metal centers as a result of Fe protein binding to the MoFe protein. The midpoint potentials for the three nitrogenase metal centers, namely the [4Fe-4S] cluster of the Fe protein, and the [8Fe-7S] (or P-) clusters and FeMo cofactors (or M-centers) of the MoFe protein, were determined within a nondissociating nitrogenase complex prepared with a site-specifically altered Fe protein (Leu at position 127 deleted, L127Delta). The midpoint potential for each metal center was determined by mediated redox titrations, with the redox state of each center being monitored by parallel and perpendicular mode EPR spectroscopy. The midpoint potential of the Fe protein [4Fe-4S]2+/1+ cluster couple was observed to change by -200 mV from -420 mV in the uncomplexed L127Delta Fe protein to -620 mV in the L127Delta Fe protein-MoFe protein complex. The midpoint potential of the two electron oxidized couple of the P-clusters (P2+/N) of the MoFe protein was observed to shift by -80 mV upon protein-protein complex formation. No significant change in the midpoint potential of an oxidized state of FeMoco (Mox/N) was observed upon complex formation. These results provide insights into the energetics of intercomponent electron transfer in nitrogenase, suggesting that the energy of protein-protein complex formation is coupled to an increase in the driving force for electron transfer. The results are interpreted in light of the expected changes in the protein environments of the metal centers within the nitrogenase complex.

Aluminum fluoride inhibition of nitrogenase: stabilization of a nucleotide.Fe-protein.MoFe-protein complex.

Coupling of ATP hydrolysis to electron transfer in nitrogenase has properties similar to nucleotide-dependent switch proteins. Aluminum fluoride, a powerful inhibitor of some switch proteins, is a progressive, slowly reversible (t1/2 for reversal > 21 h) inhibitor of nitrogenase that requires both component proteins (Fe-protein and MoFe-protein) and nucleotide (either ATP or ADP). The pseudo first-order inhibition is dependent on the aluminum fluoride species, AlF4, and is linear with [Al] concentration (nonsaturating) at a pH optimum near 7.1-7.3. The inhibitor appears to react with the transient complex of the two component proteins and nucleotide. Although ADP can support the AlF inhibition, the rate of inhibition is more than 30-fold greater with ATP, which suggests the reactive conformation more closely resembles ATP hydrolysis. Conditions that increase enzymic turnover (protein concentration and component ratio) also increase the rate of inhibition, while ionic strength which slows enzymic activity spares the inhibition. The inhibited protein was isolated by gel filtration chromatography and found to be an AlF4-ADP-Fe-protein.MoFe-protein complex with the ratio of 2:1 that is consistent with two active sites per MoFe-protein alpha 2 beta 2 tetramer. Hence, inhibition by AlF4 is the stabilization of a complex that no longer hydrolyzes ATP or reduces substrates. We propose that AlF-ADP is tightly bound only in Fe-protein conformations obtained in the complex with MoFe-protein. Ligands (including Arg-46) at the base of a flexible flap on the Fe-protein could immobilize MoFe-protein--Fe-protein interface, thereby preventing dissociation of the complex.

Nucleotide-iron-sulfur cluster signal transduction in the nitrogenase iron-protein: the role of Asp125.

Electron transfer in nitrogenase involves a gating process initiated by MgATP (magnesium adenosine triphosphate) binding to Fe-protein. The redox site, an 4Fe:4S cluster, is structurally separated from the MgATP binding site. For MgATP hydrolysis to be coupled to electron transfer, a signal transduction mechanism is proposed that is similar to that in guanosine triphosphatase proteins. Based on the three-dimensional structure of Fe-protein, Asp125 is likely to be part of a putative transduction path. Altered Fe-protein with Glu replacing Asp has been prepared and retains the ability for the initial nucleotide-dependent conformational change. However, either MgADP or MgATP can induce the shift and Mg binding to the nucleotide is no longer essential.

The Coupling of Electron Transfer in Nitrogenase to the Hydrolysis of Magnesium Adenosine Triphosphate

The Coupling of Electron Transfer in Nitrogenase to the Hydrolysis of Magnesium Adenosine Triphosphate