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New Mechanistic Insights about the Enzyme Cytochrome c nitrite reductase (ccNiR) from studies of the wild type and its variants
Author(s) -
Shahid Shahid,
Pacheco A. Andrew
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04735
Subject(s) - chemistry , nitrite reductase , nitrite , active site , moiety , stereochemistry , reductase , periplasmic space , heme , titration , electron transport chain , photochemistry , catalysis , electron transfer , redox , enzyme , inorganic chemistry , biochemistry , organic chemistry , escherichia coli , nitrate , gene
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, in vitro we have found it possible to trap putative intermediates, or to release partially reduced nitrogen species such as nitric oxide, by controlling the electrochemical potential at which reduction takes place. This poster will present UV‐Vis spectropotentiometric titrations of the active site variants R103Q and H257Q, and steady‐state and stopped‐flow kinetic studies of ccNiR‐catalyzed reduction of nitrite to nitric oxide by the weak reductant N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD). Wild type nitrite‐loaded ccNiR is reduced chemically or electrochemically at very high potentials (midpoint potential of 246 mV vs SHE) in a concerted 2‐electron process that forms an {FeNO}7 moiety at the active site. By contrast, the H257Q variant’s active site midpoint potential is downshifted to 125 mV vs SHE and is now a 1‐electron reduction to yield an {FeNO}6 moiety. The wild type catalyzes the reduction of nitrite to NO • by TMPD. H257Q does not appear to catalyze this reaction, which may be due to its lower active site midpoint potential or to the much slower turnover of the mutant; studies with more powerful reductants are now under way.