Demonstration of proton-coupled electron transfer in the copper-containing nitrite reductases.
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 approximately 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 >10 mM. 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 approximately 0.61 +/- 0.02 between 3 and 10 mM. 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.
Brenner
Sibylle
S
Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, United Kingdom.
Heyes
Derren J
DJ
Hay
Sam
S
Hough
Michael A
MA
Eady
Robert R
RR
Hasnain
S Samar
SS
Scrutton
Nigel S
NS
D000421:Alcaligenes / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry / Q000378:metabolism; D004583:Electrons; D006863:Hydrogen-Ion Concentration; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry* / Q000378:metabolism; D011522:Protons
D001426:Bacterial Proteins; D009573:Nitrites; D011522:Protons; D009569:Nitric Oxide; D003300:Copper; D009572:Nitrite Reductases; D017666:Deuterium Oxide