Evidence for Functionally Relevant Encounter Complexes in Nitrogenase Catalysis

Abstract

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.

Nitrogenase-mimic iron-containing chalcogels for photochemical reduction of dinitrogen to ammonia

“…Electron transfer during this catalytic process is believed to proceed from a [4Fe:4S] cluster located in the Fe protein to another Fe/S cluster (the P cluster) buried in the MoFe protein and finally to the FeMoco (Fig. 1A) (2,5,6). Whereas the role of Mo in the reactivity of nitrogenase has been the subject of long debate, iron is now well recognized as the only transition metal essential to all nitrogenases, and recent biochemical and spectroscopic data point to iron as the site of N 2 binding in the FeMoco (7)(8)(9).…”

Determination of nucleoside triphosphatase activities from measurement of true inorganic phosphate in the presence of labile phosphate compounds

“…Nitrogenase component proteins were purified from Azotobacter vinelandii as described previously [9]. The FeP and MoFeP had specific activities of 1600–2000 and 2200–2600 nmol C 2 H 4 min −1 mg −1 protein, respectively.…”

“…The enzymatic reactions were initiated by the addition of FeP and terminated with 0.30 mL of 5 M NaCl after 10 minutes. Protein concentrations were determined via Fe chelation in 6.4 M guanidine hydrochloride by 2,2-bipyridine using an extinction coefficient of 8650 M −1 cm −1 at 522 nm [9]. …”

“…Our interest in developing an improved assay for nucleotide hydrolysis is based on our work with nitrogenase [8,9], which catalyzes such a complex, ATP-driven biochemical reaction, namely the reduction of atmospheric N 2 into NH 3 [10]: $${\mathrm{N}}_2+8{\mathrm{e}}^{-}+8{\mathrm{H}}^{+}+16\text{ATP}\to 2{\text{NH}}_3+{\mathrm{H}}_2+16\text{ADP}+16{\mathrm{P}}_i$$…”

“…Furthermore, if the Ca 2+ precipitation strategy is to be utilized, ethylenediaminetetraacetic acid (EDTA), since it chelates Ca 2+ , cannot be used to quench enzyme activity, nor can sodium dodecyl sulfate (SDS) [28], as it interferes with subsequent phosphomolybdate complex formation. However, substrate reduction by nitrogenase depends on electrostatic interactions between FeP and MoFeP to facilitate each ET cycle [9], and a large enough excess of salt ions fully inhibits activity [34]. As many other NTPase activities involve electrostatic interactions with protein partners [35,36], we reasoned that salt, rather than acid, can be used as a chemically mild quencher of NTPase activities.…”