Electrocatalytic Nitrite Reduction by a Monomeric NrfA: Commonality in Ammonification Mechanisms.

Abstract

Cytochrome c nitrite reductase (NrfA) is a pentaheme enzyme capable of the six-electron reduction of nitrite to ammonia, which is a key step in the nitrogen cycle. All NrfA enzymes appear to have a branched set of two heme-based pathways for electron transfer to a conserved active site, and until recently, NrfA enzymes from a variety of microorganisms were considered to possess a homodimeric structure; yet, recent efforts have shown that in solution, purified Geobacter lovleyi (Gl) NrfA is a monomer. Direct protein electrochemistry has been used in the past to characterize the dimeric NrfAs from Escherichia coli and Shewanella oneidensis, revealing features of maximal activity as a function of nitrite concentration, and redox poise, both of which were interpreted in terms of the dimeric structure providing multiple redox equivalents. Here, we examine Gl NrfA using protein film electrochemistry and find that all of the features that were associated with the dimeric enzymes are also found in the monomeric enzyme. Further, we probe the contribution of specific heme environments through investigation of two His to Met heme ligand mutants, each along a different branch of the electron transfer network, which demonstrates that each path is likely essential to support native-like catalysis.