Spectroelectrochemical Investigation of Intramolecular and Interfacial Electron-Transfer Rates Reveals Differences Between Nitrite Reductase at Rest and During Turnover

Abstract

A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Förster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (≥520 μM), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme's active site, gating the intramolecular electron transfer.

Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase

“…Several pulse radiolysis162122, protein-film voltammetry919 and laser-flash-photolysis studies57 of different CuNiRs have shown that during single turnover experiments, the rate of inter-Cu ET is decreased by the presence of nitrite. The degree of inhibition is pH-dependent and for wild-type Ax NiR at pH 7 the k obs decreases from 370 to ~250 s −1 at saturating nitrite concentrations57.…”

“…It has recently been shown that protons, in addition to being a substrate in this reaction, are involved in controlling the rate of electron transfer (ET) between the type-1 Cu (T1Cu) centre and the catalytic type-2 Cu (T2Cu) centre of the enzyme56789.…”

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

“…Extensive spectroscopic and structural studies of CuNiRs (Strange et al, 1999;Boulanger et al, 2000;Boulanger & Murphy, 2001;Tocheva et al, 2004Tocheva et al, , 2007Antonyuk et al, 2005;Krzemiń ski et al, 2011;Wijma et al, 2006;Hough et al, 2008;Leferink et al, 2011Leferink et al, , 2012 have suggested a general catalytic mechanism in which bound water in the resting state is replaced by nitrite, and internal electron transfer occurs between the T1Cu and T2Cu sites. Two protons are required for the reaction, which are thought to be provided by Asp98 (Asp CAT ) and His255 (His CAT ) via a bridging water molecule, leading to cleavage of the bound NO 2 À to produce NO and H 2 O. Conformational rearrangements of Asp CAT have been proposed to be important in substrate guidance and proton transfer (Antonyuk et al, 2005), while His CAT may respond to the redox state.…”

Copper‐Containing Nitrite Reductase Employing Proton‐Coupled Spin‐Exchanged Electron‐Transfer and Multiproton Synchronized Transfer to Reduce Nitrite

“…On the other hand, at high pH or high nitrite concentrations, the binding of nitrite to oxidized T2Cu occurs prior to ET. [24,39,40] Additionally,t he reactionm echanisms of CuNiRs have been discussed as ar esult of many theoretical studies. [24,39,40] Additionally,t he reactionm echanisms of CuNiRs have been discussed as ar esult of many theoretical studies.…”