Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen.
The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.
Flock
Ulrika
U
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
Watmough
Nicholas J
NJ
Adelroth
Pia
P
D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D006418:Heme / Q000378:metabolism; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010101:Oxygen Consumption; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010782:Photolysis; D011522:Protons; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
D003574:Cytochrome c Group; D008024:Ligands; D016877:Oxidants; D011522:Protons; D014867:Water; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D010100:Oxygen