A Reduced F₄₂₀-Dependent Nitrite Reductase in an Anaerobic Methanotrophic Archaeon

Abstract

Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F₄₂₀-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F₄₂₀ (F₄₂₀H₂), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F₄₂₀H₂. Instead, it acted as an F₄₂₀H₂-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 μM; F₄₂₀H₂, 14 μM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F₄₂₀H₂-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E⁰′ [standard potential], +440 mV) but not sulfite (E⁰′, −116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies.

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