Probing the All-Ferrous States of Methanogen Nitrogenase Iron Proteins.

The Fe protein of nitrogenase reduces two C1 substrates, CO and CO, under ambient conditions when its [FeS] cluster adopts the all-ferrous [FeS] state. Here, we show disparate reactivities of the - and -encoded Fe proteins from (designated NifH and VnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO 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 VnfH adopts the all-ferrous state at a more positive reduction potential than NifH, which could account for the weaker reactivity of the VnfH toward C1 substrates than NifH.

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN.

NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli.