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Demonstration of proton-coupled electron transfer in the copper-containing nitrite reductases

Brenner, S and Heyes, DJ and Hay, S and Hough, MA and Eady, RR and Hasnain, SS and Scrutton, NS (2009) 'Demonstration of proton-coupled electron transfer in the copper-containing nitrite reductases.' Journal of Biological Chemistry, 284 (38). 25973 - 25983. ISSN 0021-9258

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Abstract

The reduction of nitrite (NO2-) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H2O and D2O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33±0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H2O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10mM. As a consequence, the observed ET process was faster in D2O than in H2O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10mM. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Item Type: Article
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Jim Jamieson
Date Deposited: 10 Nov 2011 15:53
Last Modified: 12 Aug 2019 13:15
URI: http://repository.essex.ac.uk/id/eprint/1314

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