Faculty Collaboration Database

Trapping of a Putative Intermediate in the Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite: Implications for the ccNiR Reaction Mechanism. J Am Chem Soc 2019 Aug 28;141(34):13358-13371

Date

08/06/2019

Pubmed ID

31381304

DOI

10.1021/jacs.9b03036

Scopus ID

2-s2.0-85071714374 (requires institutional sign-in at Scopus site)   23 Citations

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.

Author List

Ali M, Stein N, Mao Y, Shahid S, Schmidt M, Bennett B, Pacheco AA

Authors

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University
Arsenio Andrew Pacheco in the Chemistry and Biochemistry department at University of Wisconsin - Milwaukee

MESH terms used to index this publication - Major topics in bold

Ammonia
Catalysis
Catalytic Domain
Cytochromes a1
Cytochromes c1
Models, Molecular
Nitrate Reductases
Nitrites
Oxidation-Reduction
Protein Conformation
Shewanella
Spectrophotometry, Ultraviolet
Substrate Specificity