Probing the All-Ferrous States of Methanogen Nitrogenase Iron Proteins

The Fe protein of nitrogenase reduces two C1 substrates, CO2 and CO, under ambient conditions when its [Fe4S4] cluster adopts the all-ferrous [Fe4S4](0) state. Here, we show disparate reactivities of the nifH- and vnf-encoded Fe proteins from Methanosarcina acetivorans (designated MaNifH and MaVnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO2 and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that MaVnfH adopts the all-ferrous state at a more positive reduction potential than MaNifH, which could account for the weaker reactivity of the MaVnfH toward C1 substrates than MaNifH. More importantly, MaVnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe4S4](0) cluster at a reduction potential of -0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of -0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe4S4](0) state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe4S4](0/2+) redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

Automated summary

The Fe proteins MaNifH and MaVnfH from Methanosarcina acetivorans exhibit different reactivities towards C1 substrates in the all-ferrous state, with MaVnfH showing weaker reactivity due to adopting the all-ferrous state at a more positive reduction potential. This suggests the possibility of MaVnfH accessing the all-ferrous state under physiological conditions, which has implications for the reaction mechanism of nitrogenase.