Trinity Hamilton

The metalloenzyme nitrogenase is utilized by microbial diazotrophs to accomplish the majority of biological nitrogen fixation. Many different forms of the enzyme nitrogenase are known to exist. They are categorized by the type of metal cluster providing the N2-binding site, which is also the active site of the enzyme. The nitrogenase best-characterized to date has an active site with the compostition [7Fe-9S-1MoX-homocitrate], owing to its designation as a MoFe-cofactor. The protein containing the active site also houses a P-cluster ([8Fe-7S]) necessary for electron transfer. A smaller, MgATP-dependent, Fe protein with a [4Fe-4S] cluster functions to deliver electrons to the active site. A variety of structural and functional studies have shed light on the mechanism of N2 reduction by nitrogenase via the MoFe protein and the Fe protein. The following will provide evidence for the role of the P-cluster in electron transfer between the Fe protein and the FeMo-cofactor through chemical and structural analysis of the metal cluster and its substrates. Electron paramagnetic resonance spectra, density functional theory, and X-ray scattering studies are just a few of the techniques utilized in the elucidation of the mechanism of the P-cluster in the Mo-dependent nitogenase. A discussion of these techniques and their role in the characterization of this metalloenzyme will elucidate the current understanding of the metal clusters and their part in N2 reduction. Introduction. The biological relevance of nitrogen cannot be overstated in that it is necessary in many biological compounds, namely amino and nucleic acids. Atmospheric nitrogen, the largest reservoir of nitrogen, is unavailable for incorporation into biomolecules. The enzyme nitrogenase is responsible for nearly all biological nitrogen fixation, in which dinitrogen is converted to ammonia. Once converted to ammonia, incorporation of nitrogen into amino acids ensues. The reaction catalyzed by nitrogenase is as follows: N2 + 8H + 8e + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Many nitrogenases are categorized based on the metals occupying the N2-binding site, which is also the active site of the enzyme. The Mo-containing nitrogenases are the best-characterized to date. The active site of the Mo-dependent nitrogenase family is housed within the large component, the MoFe protein. It contains a metal cluster known as the FeMo-cofactor, [7Fe-9S-1Mo-X-homocitrate] and the P-cluster. A small Fe protein with a [4Fe-4S] cluster is responsible for supplying the active site with electrons as well as the ATP necessary to break the dinitrogen triple bond. The mechanism of electron transfer by the nitrogenase enzyme has been studied to great length. However, the debate continues over the exact path of electrons through the complex. This paper will recount the current understanding of electron transfer from the Fe protein to the FeMo-cofactor via the P-cluster. Evidence will be provided from EPR and ENDOR data as well as X-Ray crystallography, Mossbauer spectroscopy and density functional theory to support the current hypothesis of the flow of electron through the P cluster in the Modependent nitrogenase. In response to the resulting hypothesis, future research will be suggested. Methods. Electron Paramagnetic Resonance (EPR) is a valuable tool for probing structures of different oxidation states, especially those with metal centers. EPR measures the absorption of a microwave radiation by paramagnetic ions or molecules in the presence of a static magnetic field. The species being monitored must have at least one unpaired electron. EPR has provided useful insight into the ground state of the metal centers in nitrogenase components. ENDOR (Electron-Nuclear Double Resonance) combines both EPR and NMR to deduce structural characteristics of complexes. ENDOR describes distance and location of atoms surrounding a paramagnetic atom. ENDOR exploits the hyperfine coupling between nuclear and electron spin, making it a more sensitive technique than EPR. Both EPR and ENDOR are valuable techniques for assigning spin states to Fe in varying oxidation states of the [Fe-S] clusters within the nitrogenase. Mossbauer spectroscopy has been useful in assigning valency to the Fe atoms in the FeMo-cofactor and the P cluster. Mossbauer spectroscopy is especially useful in conjunction with EPR and ENDOR because of its ability to measure isomer shifts in diamagnetic species. Mossbauer spectroscopy exploits the isomer shift between Fe and Fe to assign oxidation states and spin states to Fe under varying redox conditions. Because the [8Fe-7S] cluster of the P-cluster contains all ferrous (Fe) in the resting state, its diamagnetic character can be probed using Mossbauer spectroscopy. This spectroscopic technique also reveals some of the local electronic field around each Fe atom. X-Ray Crystallography is an invaluable technique for determining structural aspects of iron-sulfur clusters. This technique has provided key insight into the spatial distribution and location of Fe, S and ligand atoms in each of the nitrogenase components. X-ray structures have provided evidence of structural changes that occur as electrons move through the nitrogenase complex. These structural changes provide direct evidence for the function of each component in the reduction of dinitrogen by the nitrogenase enzyme. Density Functional Theory (DFT) is another tool for probing the ground state of metals. It is a complex evaluation of the ground state wave functions of antiferromagnetically coupled multi-spin systems. DFT has been utilized to determine redox potentials (Em values) for redox pairs isolated by X-ray crystallography or detected by EPR, ENDOR, and Mossbauer spectroscopy. Results & Discussion. The first step in the nitrogenase mechanism occurs when the Fe protein, bound to MgATP, docks to the MoFe protein, which is a tetramer of two αβ-units. An electron is transferred from the Fe protein to the MoFe protein in conjunction with MgATP hydrolysis. The MoFe protein apparently catalyzes the hydrolysis of MgATP, which is linked to the electron transfer event. However, the two processes are not coupled and can be shown to occur independently. Once the electron transfer has been accomplished, the Fe protein dissociates from the MoFe protein, with reassociation occurring for each sequential transfer of a single electron. (1) The Fe protein is required to deliver electrons to the MoFe protein, while the actual reduction of dinitrogen takes place within the MoFe protein. The figures shown below show the αβ-tetramer of the MoFe proteins with the P-clusters and the FeMocofactors. The P-clusters are located near the Fe protein binding sites at the interface of each αβ-subunit of the MoFe protein tetramer. This suggests electrons are transferred first to the P-cluster and eventually onto the FeMo-cofactor, where the actual reduction occurs. The P-cluster bridges the interface between the αand βsubunits of the MoFe protein. The FeMo-cofactors both lie deep within the protein in the α-subunits, allowing for only small molecules to reach the active site, such as dinitrogen. (8) Figure 1. MoFe protein with the Fe proteins bound at each end of the protein. The Pcluster is located at the αβinterface of the MoFe protein. The reduction of dinitrogen is occurring at the active site, the FeMo cofactor. (5) Figure 2. Panel (a) represents the Fe protein docked at the αβ-interface of one of the MoFe protein subunits. (The α-subunit is in red and the β-subunit in blue. Panel (b) represents the relative positions of the iron-sulfur clusters in each component of the nitrogenase and the hypothetical electron-transfer between each component. (6) The P-cluster has been implicated as an electron transfer intermediate between the Fe protein and the active site. Mossbauer spectroscopy first identified the total number of Fe atoms in the P-cluster. X-ray structures and EPR analysis have determined that the P-cluster is a unique [8Fe-7S] metal cluster. The two [4Fe-4S] and [4Fe-3S] clusters share a μ6-sulfide that bridges the two clusters together when the P cluster is in its fully reduced form. In addition to the sulfide, the two iron-sulfur cubanes of the P-cluster are also bridged by two thiolates from Cys residues 88 and 95. 88 lies in the α-subunit while the 95 residue is in the β-subunit of the MeFo protein. Upon oxidation, the bridging sulfide detaches from 2 Fe proteins. (1-3,5,8) The complete [4Fe-4S] cluster is anchored in the α-subunit while the [4Fe-3S] cluster is associated mainly with the β-subunit. The P-cluster is unique in that it is the first known iron-sulfur cluster with naturally occurring amide and serine ligands in addition to cysteine ligands. The amide and serine ligands are not only exchangeable ligands, but are also protonatable. The protonatable nature of these ligands would make the P-cluster sensitive to local pH changes and explain resulting structural changes although this theory has yet to be supported. (6) Structural studies of the P-cluster in oxidized and reduced forms have revealed significant changes occurring between the two states. Most notably, the bridging sulfide apparent in the reduced form (P or b in the figures) detaches from the 2 irons in the oxidized form. The P-cluster in the oxidized form consists of a [4Fe-4S] cluster lying primarily in the α-subunit of the MoFe protein and a [4Fe-3S] cluster that is primarily in the β-subunit. The hypothesis for this structural change is that the P-cluster also participates in protonation/deprotonation reactions. This hypothesis was supported by Em values for redox couple P which were shown to vary linearly over a 6.0-8.4 pH range. Changing pH did not affect other redox pairs, indicating the +1 to +2 redox change must involve the release of a proton. Furthermore, the P-cluster may function to couple proton and electron transfer. (1,5) Figure 3. Structural representation of the oxidized (b) and reduced forms (a) of the the P-cluster. The green atoms represent Fe, while the yellow are S atoms. In the oxidized state, the P-cluster is no longer bridged by the sulfur atom. In addition, two of the cluster Fe atoms undergo significant structural and spatial changes. (6) Figure 4. Schematic representation of the oxidation states of the P-cluster. P represents the reduced form with the sulfide bridge intact. The two bridging Cys residues are also shown. The 2-electron oxidized form is P, in which the sulfide bridge is no longer intact. (2) The current hypothesis is that the P-cluster receives electrons from the Fe protein; however, the oxidation state of an electron-accepting P-cluster has yet to be determined. It is thought that either there is a further reduced state of the P cluster that has yet to be detected or that the P-cluster first sends electrons to the FeMo-cofactor, becoming oxidized and then accepting electrons from the Fe protein. Figure 5. A schematic representation of the postulated electron transfer from the Fe protein to the Pcluster. (6) The oxidation states of the [8Fe-7S] cluster have been studied quite extensively. EPR, ENDOR and Mossbauer spectroscopy in conjunction with the aforementioned Xray analysis have been able to identify redox changes in the Fe atoms of the P-cluster. By tracking the changes in spin states and oxidation states of the Fe atoms, redox reactions of the electron transfer through the P-cluster have been postulated. For example, when the MoFe protein is in its fully oxidized state, the P cluster is found to be reduced and appears as a diamagnetic species, with S=0 and all the Fe atoms in the ferrous state. The P-cluster can then be artificially oxidized to several states. The one-electron oxidized state gives rise to a mixed spin state of 1/2 and 5/2 with a perpendicular mode EPR signals with g tensor values of g=2 and g=5-8. A proposed model of the P state is that it may be only partially oxidized, giving rise to the mixed population of spin states. The P state of the cluster gives rise to a parallel mode EPR signal at g=11.8, indicative of spin coupling within the cluster upon oxidation of both halves of the P-cluster. Using DFT, the Em values of the first two oxidation states were found to be -380mV. As mentioned previously, the degeneracy of these oxidation states is lost at varying pH values. The spin state of this oxidized P-cluster is S≥3. The P oxidation state is essentially irreversible with spin states of 1/2 and 7/2. (4) The current belief is that the P-cluster channels electrons from the Fe protein to the FeMoco, which is the active site of the enzyme. The cofactor is the site where the actual reduction of dinitrogen occurs. From the position of the P-cluster, it is assumed that it acts as an intermediate in the electron transfer pathway between the Fe protein and the FeMoco. However, only computational evidence of this pathway has been found. Figure 6. Hypothetical electron transfer pathway from the Pcluster to the FeMo-co shown by the dashed line, calculated using HARLEM. The calculations identified the dashed line as the single favored pathway for electron transfer.