The reaction of Pseudomonas nitrite reductase and nitrite. A stopped-flow and EPR study.
The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy. The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2). [formula: see text] The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1. The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O. Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551. When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.
Silvestrini
M C
MC
Department of Biomedical Sciences, University of L'Aquila, Italy.
Tordi
M G
MG
Musci
G
G
Brunori
M
M
D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008962:Models, Theoretical; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D013997:Time Factors
D009573:Nitrites; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases