Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass

Julius Campeciño, Satyanarayana Lagishetty, Zdzislaw Wawrzak, Victor Sosa Alfaro, Nicolai Lehnert, Gemma Reguera, Jian Hu, Eric L. Hegg*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca21-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca21-dependent enzymes, suggesting convergent evolution.

Original languageEnglish (US)
Pages (from-to)11455-11465
Number of pages11
JournalJournal of Biological Chemistry
Volume295
Issue number33
DOIs
StatePublished - Aug 14 2020

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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