The P-cluster is a unique iron-sulfur center that likely functions as a dynamic electron (e−) relay site between the Fe-protein and the catalytic FeMo-cofactor in nitrogenase. The P-cluster has been shown to undergo large conformational changes upon 2-e− oxidation which entail the coordination of two of the Fe centers to a Ser side chain and a backbone amide nitrogen, respectively. Yet, how and if this 2-e− oxidized state (POX) is involved in catalysis by nitrogenase is not well established. Here, we present the crystal structures of reduced and oxidized MoFe-protein (MoFeP) from Gluconacetobacter diazotrophicus (Gd), which natively possesses an Ala residue in the position of the Ser ligand to the P-cluster. While reduced Gd-MoFeP is structurally identical to previously characterized counterparts around the FeMo-cofactor, oxidized Gd-MoFeP features an unusual Tyr coordination to its P-cluster along with ligation by a backbone amide nitrogen. EPR analysis of the oxidized Gd-MoFeP P-cluster confirmed that it is a 2-e− oxidized, integer-spin species. Importantly, we have found that the sequence positions corresponding to the Ser and Tyr ligands are almost completely covariant among Group I nitrogenases. These findings strongly support the possibility that the POX state is functionally relevant in nitrogenase catalysis and that a hard, O-based anionic ligand serves to stabilize this state in a switchable fashion.

Nitrogenase is the only enzyme that can convert atmospheric dinitrogen (N2) into the biologically usable ammonia. To achieve this multi-electron redox process, the nitrogenase component proteins, MoFe-protein (MoFeP) and Fe-protein (FeP), repeatedly associate and dissociate in an ATP dependent manner, where one electron is transferred from FeP to MoFeP per association. Here, we provide evidence for the first time that encounter complexes between FeP and MoFeP play a functional role in nitrogenase turnover. The encounter complexes are stabilized by electrostatic interactions involving a positively charged patch on the β-subunit of MoFeP. Three single mutations (βAsn399Glu, βLys400Glu, and βArg401Glu) in this patch were generated in Azotobacter vinelandii MoFeP. All of the resulting variants displayed decreases in specific catalytic activity, with the βK400E mutation showing the largest effect. As simulated by the Thorneley-Lowe kinetic scheme, this single mutation lowered the rate constant for FeP-MoFeP association five-fold. We also found that the βK400E mutation did not affect the coupling of ATP hydrolysis with electron transfer (ET) between FeP and MoFeP. These data suggest a mechanism where FeP initially forms encounter complexes on the MoFeP β-subunit surface en route to the ATP-activated, ET-competent complex over the αβ-interface.

In this review, we summarize our recent efforts toward understanding electron transfer (ET) processes in nitrogenase, the only enzyme capable of reducing dinitrogen to ammonia. We discuss new structural and biochemical perspectives on the role of ATP‐dependent interactions between the two components of nitrogenase, Fe‐protein (FeP) and MoFe‐protein (MoFeP), and how these interactions may regulate interprotein ET and catalysis. We also discuss the implications of our work on FeP‐ and ATP‐independent, photoredox‐activated substrate reduction by MoFeP. Elucidating why and how ATP‐hydrolysis is needed to control electron and proton flow in nitrogenase is not only a fundamentally important question in biological redox chemistry and energy transduction, but it also holds the key to understanding the intimate mechanism of dinitrogen reduction.

One of the most common assays for NTPase activity entails the quantification of inorganic phosphate (Pi) as a colored phosphomolybdate complex at low pH. While this assay is very sensitive, it is not selective for Pi in the presence of labile organic phosphate compounds (OPCs). Since NTPase activity assays typically require a large excess of OPCs, such as nucleotides, selectivity for Pi in the presence of OPCs is often critical in evaluating enzyme activity. Here we present an improved method for the measurement of enzymatic nucleotide hydrolysis as Pi released, which achieves selectivity for Pi in the presence of OPCs while also avoiding the costs and hazards inherent in other methods for measuring nucleotide hydrolysis. We apply this method to the measurement of ATP hydrolysis by nitrogenase and GTP hydrolysis by elongation factor G (EF-G) in order to demonstrate the broad applicability of our method for the determination of nucleotide hydrolysis in the presence of interfering OPCs.