Electron/Proton Coupling in Bacterial Nitric Oxide Reductase during Reduction of Oxygen
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- 作者：Ulrika Flock ; Nicholas J. Watmough ; and Pia Ä ; delroth
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 9, 2005
- 年：2005
- 卷：44
- 期：31
- 页码：10711 - 10719
- 全文大小：234K
- 年卷期：v.44,no.31(August 9, 2005)
- ISSN：1520-4995
文摘
The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N2O (2NO + 2H+ + 2e- 
N2O + H2O), which is an obligatory step in thesequential 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 b3, and a non-heme iron FeB, and belongs tosame superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electronacceptor; 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 initiatedusing the flow-flash technique, and the progress of the reaction monitored by time-resolved opticalabsorption spectroscopy. Two major phases with time constants of 40 
s and 25 ms (pH 7.5, 1 mM O2)were observed. The rate constant for the faster process was dependent on the O2 concentration and isassigned to O2 binding to heme b3 at a bimolecular rate constant of 2 × 107 M-1 s-1. The second phase(
= 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protonsfrom the bulk solution. The rate constant for this phase shows a pH dependence consistent with ratelimitation by proton transfer from an internal group with a pKa = 6.6. This group is presumably an aminoacid residue that is crucial for proton transfer to the catalytic site also during NO reduction.
N2O + H2O), which is an obligatory step in thesequential 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 b3, and a non-heme iron FeB, and belongs tosame superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electronacceptor; 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 initiatedusing the flow-flash technique, and the progress of the reaction monitored by time-resolved opticalabsorption spectroscopy. Two major phases with time constants of 40 s and 25 ms (pH 7.5, 1 mM O2)were observed. The rate constant for the faster process was dependent on the O2 concentration and isassigned to O2 binding to heme b3 at a bimolecular rate constant of 2 × 107 M-1 s-1. The second phase( = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protonsfrom the bulk solution. The rate constant for this phase shows a pH dependence consistent with ratelimitation by proton transfer from an internal group with a pKa = 6.6. This group is presumably an aminoacid residue that is crucial for proton transfer to the catalytic site also during NO reduction.