Protein Film Voltammetry of Copper-Containing Nitrite Reductase Reveals Reversible Inactivation

Abstract

The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of approximately 0.1 s(-1). The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

“…5A, by using the scheme in Fig. 6A the dwell time distributions can be understood reasonably well with only two forward ET rate constants, one exhibited without bound substrate, k C , and one exhibited with bound substrate, k D (21,32). As shown in Fig.…”

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

“…27, the substrate affinity of the T1Cu-reduced enzyme species is greater than that of the completely oxidized enzyme. The inter-copper ET observed in the presence of nitrite may therefore include varying contributions from all three reaction sequences labeled 1-3 in Scheme 2 depending on the substrate concentration 5 (14,16,19,26,27,33,46). For several reasons this mechanism is difficult to model mathematically: (i) there may be a pre-equilibrium yielding (at least) two initial enzyme species; (ii) laser experiments are treated as equilibrium perturbation experiments (7,20,30) resulting in complex expressions for multistep reactions (67); (iii) reaction branching is likely to occur after the T1Cu reduction, i.e.…”

“…In the absence of substrate, the difference in the redox potentials has been found to be insignificant at pH 7 (EЈ 0 (T1Cu) ϳ240 mV and EЈ 0 (T2Cu) ϳ230 mV (20)), implying a thermodynamically equal electron distribution between the two metal centers. From an enzymatic point of view, however, approaching this equilibrium position on such a fast time scale (Ն150 s Ϫ1 ) is unfavorable in the absence of substrate, as NiR has been shown to form an inactive species with a reduced T2Cu that is devoid of the H 2 O/OH Ϫ ligand and unable to bind nitrite (26,27). Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7,16,25,(27)(28)(29)(30).…”

“…Moreover, the presence of nitrite has been postulated to be relayed to the T1Cu site via the so-called substrate sensor loop (via His-94, Asp-92, and His-89 in AxNiR), thereby triggering ET to the T2Cu (19,27,29,32). The tight coupling of ET to the presence of substrate has been argued to prevent the formation of a deactivated enzyme species with a prematurely reduced T2Cu (14,16,19,26,27,33). In accordance with such a feedback mechanism, in a combined crystallographic and single-crystal spectroscopic study, inter-copper ET could only be detected in crystals where nitrite was bound to the T2Cu site, whereas in the absence of substrate no such ET was observed (34).…”

Protein film voltammetry: electrochemical enzymatic spectroscopy. A review on recent progress

“…The movement of the electrons between the electronic conductor (electrode) and the active center of the redox-active protein is instrumentally detected as a currentpotential profile having specific features depending on the measuring time and the applied potential. From the features of the voltammetric outputs, one can obtain information about the thermodynamic and kinetic parameters of the electron transfer process between the electrode and the redox sites of the proteins [1,4] and in addition, an insight into the mechanism of protein interactions with the given substrates [5][6][7][8][9][10]. To do this, a good theoretical background of the ongoing electrode mechanism is required.…”