Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase
2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, 25531-25537 p.Article in journal (Refereed) Published
Nitric-oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) (2NO + 2H(+) + 2e(-) -> N2O + H2O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe-B. Here, we have studied the reaction between fully reduced NOR and NO using the ""flow-flash"" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of similar to 1 mu s at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to FeB before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 mu M NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.
Place, publisher, year, edition, pages
2010. Vol. 285, no 33, 25531-25537 p.
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:su:diva-50090DOI: 10.1074/jbc.M110.123984ISI: 000280682400047OAI: oai:DiVA.org:su-50090DiVA: diva2:382297
authorCount :52010-12-302010-12-212015-10-19Bibliographically approved