Six-electron Reduction Of Nitrite Research Articles

Redox Enzymology of Shewanella Oneidensis Cytochrome C Nitrite Reductase

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has previously been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) adsorbed to a graphite electrode, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

Electron transport-linked proton translocation at nitrite reduction in Campylobacter sputorum subspecies bubulus.

Campylobacter sputorum subspecies bubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate and L-lactate are used as hydrogen donors. Cells of C. sputorum grown with nitrate or nitrite contain cytochromes of the b- and c-type and a carbon monoxide-binding cytochrome c. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate or L-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. Leads to H+/O values and leads to H+/NO-2 values were measured with ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) and L-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a leads to H+/2e value of 2.0. The leads to H+/O and leads to H+/NO-2 values predicted by this scheme are in good agreement with the experimental values.