Trapping of a Putative Intermediate in the Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite: Implications for the ccNiR Reaction Mechanism

Abstract

Cytochrome c nitrite reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-electron step to generate an {FeNO} 7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of +20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO} 7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO} 7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least −120 mV, well below the midpoint potential for {FeNO} 7 formation. Thus, it appears that an {FeNO} 7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

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

“…The UV-Vis and EPR spectra (Fig. S2 and S3) are consistent with previously characterized NrfA enzymes (10,(22)(23)(24).…”

The role of porphyrin peripheral substituents in determining the reactivities of ferrous nitrosyl species

“…The rst PCET forms a {FeNO} 7 species (with protonation of Arg 114 residue) which subsequently generates {FeHNO} 8 species through another PCET process, releasing only a trace amount of the {FeNO} 7 intermediate. 11,67 In contrast, Cd 1 NiR undergoes an electron transfer (ET) from cytochrome c, forming the {FeNO} 7 intermediate, which releases NO rapidly (k off $ 200 s À1 ). 13,19 There is an extensive debate on whether the NO is released from 69 suggested that maximum ruffling was present in {FeTPC-NO} 7 species, which possess a chlorin ring (see the ESI, Section 9 †), and had a relatively strong Fe-NO bond.…”

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…Ferrous heme–nitrosyl complexes, ls-{FeNO} 7 in the Enemark–Feltham notation, have been extensively studied with many spectroscopic and theoretical methods as described in Section . Most recently, Pacheco and coworker were able to trap a ls-{FeNO} 7 intermediate in S. oneidensis NrfA, using spectro­potentiometry . Previously, it had been shown by protein film voltammetry experiments that catalytic turnover in S. oneidensis NrfA requires an applied potential of at least −120 mV (vs SHE), which lies below the midpoint potential for the formation of the ls-{FeNO} 7 complex. , This indicates that it might be possible to trap the ls-{FeNO} 7 intermediate if a more positive potential is applied that stalls the catalytic cycle, thus preventing ammonia formation.…”

“…c nitrite reductases (C c NIRs), following the reaction: The active site heme of C c NIRs is unusual, as it shows axial Lys coordination, although the significance of the proximal Lys for catalysis is unclear. Notably, nitrite reduction to ammonia by C c NIRs is proposed (from computational studies) to involve the complete set of heme-based ls-{FeNO} 6−8 intermediates, but details of the mechanism and experimental characterization of such reaction intermediates are lacking . The DNRA pathway is particularly interesting as a natural mechanism to convert nitrite and nitrate back into ammonium (i.e., fertilizer), instead of converting them into gaseous products and losing nitrogen from the soil …”