Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

Abstract

The reduction of nitrite (NO 2 ؊ ) 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 H 2 O and D 2 O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ϳ1.33 ؎ 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substratefree AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H 2 O 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 D 2 O than in H 2 O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ϳ0.61 ؎ 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.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

“…Instead, Kobayashi et al (15) proposed that reduction-induced structural change of His255 is responsible for the gating-like mechanism. Because it has been recently proven that intramolecular ET in CuNiR is accompanied by PT and hence proton-coupled ET (PCET) (17,18), one can readily speculate that intramolecular ET contributes PT to NO 2 − and that the structural change of His255 is involved in PCET. Crystal structures of CuNiR from Rhodobacter sphaeroides (RhsNiR) implies this possibility because His287 in RhsNiR, which corresponds to His255, seems to show pH-and redox-dependent conformational changes (19,20).…”

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

“…A combination of new EIGER detector technology for rapid data collection (Casanas et al, 2016) and more streamlined MSOX data collection with minimal dead time presents an exciting avenue for further development of this technique. This provides a parallel approach to XFEL-based SFX and serial femtosecond rotation crystallography (SF-ROX), where radiation damage-free structures are achieved but where electrons to drive the reaction could potentially be provided by incorporating NADH and activating using an Nd/YAG laser, as we have previously performed for solution experiments (Brenner et al, 2009).…”

Fluorescence Lifetime Analysis of Nitrite Reductase from Alcaligenes xylosoxidans at the Single‐Molecule Level Reveals the Enzyme Mechanism

“…[17] This mechanism later received further support from the work of Scrutton and co-workers. [18] Herein we report evidence from single-molecule FLIM studies to shed new light on the catalytic mechanism of NiRs. For our experiments the enzyme was covalently labeled with a fluorescent molecule with an emission spectrum that overlaps with the absorption spectrum of the oxidized T1 copper site ( Figure S4 in the Supporting Information).…”