Characterization of the “iron only” nitrogenase from Rhodobacter capsulatus

Mechanism of N2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H2

“…This assumption was later challenged by the demonstration of N 2 fixation by bacterial strains with deletions of the Mo-nitrogenase genes and cells grown in Mo-deficient media. 46–48 However, doubts about Mo-independent N 2 reduction remained until purified V- and Fe-nitrogenase 24,26,49–51 systems with very little Mo content were shown to be capable of N 2 fixation, solidifying that Mo was not essential for N 2 reduction in nitrogenase. As reported here, highly purified FeFe protein from A. vinelandii has Mo and V below the detection limit of our plasma emission method, placing the Mo and V content below 0.07 mol Mo or 0.005 mol V/mol FeFe protein.…”

Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases

“…Rc AnfDGK has also been shown to feature analogous inhibition of substrate reduction as the MoFe and VFe proteins, particularly at lower partial pressures of CO . However, acetylene reduction by AnfDGK in the presence of high concentrations of CO produces an amount of ethane that exceeds ethylene production by ∼80%, whereas in V-nitrogenase, the two products formed are inhibited with similar behavior . As mentioned, hydrogen production by the MoFe and Ac VFe proteins is unsurprisingly stable in a CO environment; however, for Av V-nitrogenase, it was reported that increasing the atmosphere from 0 to 100% CO (while balancing the pressure with Ar) resulted in a decrease in the specific activity for proton reduction of ∼75% .…”

“…143 It was observed that for acetylene reduction by Av VnfDGK in the presence of CO, low electron flux (1:5, VnfH:VnfDGK) led to an increase in ethylene formation relative to higher flux (8:1) conditions, and further, ethane formation was enhanced at low pressures of CO. 236 This seems to be a unique feature of the A. vinelandii system, as the VFe protein from A. chroococcum demonstrates more typical inhibition of acetylene reduction in the presence of CO. 217 AnfDGK has also been shown to feature analogous inhibition of substrate reduction as the MoFe and VFe proteins, particularly at lower partial pressures of CO. 115 However, acetylene reduction by AnfDGK in the presence of high concentrations of CO produces an amount of ethane that exceeds ethylene production by ∼80%, whereas in V-nitrogenase, the two products formed are inhibited with similar behavior. 174 As mentioned, hydrogen production by the MoFe and Ac VFe proteins is unsurprisingly stable in a CO environment; 217 however, for Av V-nitrogenase, it was reported that increasing the atmosphere from 0 to 100% CO (while balancing the pressure with Ar) resulted in a decrease in the specific activity for proton reduction of ∼75%. 149 It is uncertain why the Av protein has this inhibition pattern, but it has been proposed that there may be two separate mechanisms for H 2 evolution, one of which is more sensitive to CO binding.…”

“…The Fe-only nitrogenase catalytic protein (AnfDGK or FeFe protein) is encoded by the anfDGK genes and has been expressed in bacterial strains that delete or inactivate the genes for Mo- and V-nitrogenases, or the cells are grown in the presence of tungstate (WO 4 2– ) to repress the expression of Mo-nitrogenase (when the organism lacks genes for VnfDGK). − The AnfDGK protein has been isolated from A. vinelandii , R. capsulatus , Rhodospirillum rubrum , and R. palustris , but the determination of the subunit composition was fraught with issues similar to those observed for V-nitrogenase. , There were initially two compositions reported for Av AnfDGK, an α 2 β 2 variant analogous to the Mo-nitrogenase, and an αβ 2 species similar to that observed for the Av VFe A protein. , Subsequent studies resulted in a composition for FeFe protein from A. vinelandii and R. capsulatus consistent with a heterohexameric α 2 β 2 δ 2 formulation and a size of M r = ∼250 kDa (AnfD ≈ 59 kDa, AnfK ≈ 51 kDa, AnfG ≈ 14 kDa). , To date, there is no reported crystal structure of AnfDGK, though one might anticipate that the protein would retain a structure analogous to those of the Mo- and V-dependent systems based on the similarity between the three systems. AnfDGK has similar subunit size and composition to V- and Mo-nitrogenases (noting that Mo-nitrogenase lacks a NifG subunit), and when the V- and Mo-nitrogenase crystal structures are overlaid, there is modest overlap, with a root–mean–squared deviation of 1.97 Å for all atoms .…”

Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

“…A more comprehensive biochemical description of the Fe nitrogenase is presented in this work which (a) allows a well-founded distinction from the conventional Mocontaining system of the same organism, (b) yields important information on the specific influence of the Fe replacing the heterometal center (Mo), on the EPR-spectroscopic properties of the cofactor-containing Rcl" protein and on the reactivity of this enzyme with substrates, and (c) provides generally a better understanding of the catalytic particularities of the Fe nitrogenase and in this context, of the structure/function interrelationships. Part of this work was presented at the Seventh International Conference on Bioinorganic Chemistry [24].…”