Simple formal kinetics for the reversible uptake of molecular hydrogen by [Ni–Fe] hydrogenase from Desulfovibrio gigas

Abstract

Enzymatic electrocatalysis, triggered and monitored by means of cyclic voltammetry, enabled us to achieve quantitative analysis of the kinetics of the hydrogenase catalyzed process, in the 7.8±10.0 pH range, in the presence of an electrochemically generated redox mediator. The quantitative analysis can be carried out by use of a quite simple SRC model. The simplicity of the SRC model is compatible with the existence of multiple redox microstates, which can be combined in a potential adjustable triangular mechanism consisting of three catalytic cycles, which are formally identical from the kinetic point of view. The steps involved in the kinetic control of the reversible process are H 2 uptake or production at the Ni±Fe catalytic site and the intermolecular electron transfer between the mediator and the distal [4Fe24S] cluster. The related rate constants have been determined. For the two accompanying intramolecular electron transfers which proceed at equilibrium, the equilibrium constants were found to be in very good agreement with previously published data.Keywords: hydrogenase; FeS cluster; catalytic mechanism; rate constant; electrochemical methods.The reversible oxidation of molecular hydrogen by hydrogenases is a central metabolic feature of several microorganisms [1]. Hydrogenase of Desulfovibrio gigas has been studied as an archetype of [Ni±Fe]hydrogenases for a long time [2±5] and the publication of the X-ray structure in 1995 strengthened the interest in this enzyme [6]. Recently, several models of the catalytic cycle have been put forward [7±14], based on numerous multidisciplinary papers reporting the identification of intermediate species involved in the catalytic process [2±5, 15±18]. From the redox point of view, the description of the enzyme is quite complex as it contains several redox centers. Crystallographic analyses showed that the active center contains two metals, nickel and iron [6,12]. Additionally, two [4Fe24S] clusters and one [3Fe24S] cluster are distributed almost along a straight line in one of the two subunits. The [3Fe24S] cluster lies in between the two [4Fe24S] clusters, one of which is located near the active center and is named proximal, whereas the other is located near the surface of the enzyme and is named distal. The observed spatial arrangement of the three Fe±S clusters suggests that they can constitute an electron transfer path between the Ni±Fe active center and the redox partner of the hydrogenase [6,19].It is now well admitted that the six main stabilized redox states exhibited by the Ni±Fe active center can be separated in two series. The first includes the Ni-A, Ni-SU and Ni-B redox states existing at relatively high redox potentials and not efficient in the catalysis of reversible H 2 oxidation. The second series includes Ni-SI(a or b), Ni-R and Ni-C which participate in the catalytic cycle at potentials close to the H 2 /H 1 potential at a given pH. All the recent models describing hydrogen activation by [Ni±Fe]hydrogenases point to a Ni-SI 3 Ni-R 3 Ni-C 3 Ni-SI ...

Direct electrochemical study of the multiple redox centers of hydrogenase from Desulfovibrio gigas

“…Although the catalytic mechanism is still under discussion, one model for the reaction is described as the H 2 fixation to the Ni(II) at the Ni-SI form, heterolytic cleavage to form a protonated intermediate, that may be a bridge hydride between the Ni and the Fe atoms, assigned as the Ni-C form [14]. Follows the subsequent production of the reduced state Ni-R and re-oxidation with regeneration of the Ni-SI, passing by the Ni-C form [15]. Another pertinent discovery is the presence of a gas channel localized from the protein surface towards the Ni atom, giving support to the idea that Ni has the main role on the catalysis mechanism [16].…”

“…Electrochemical techniques are important tools to understand enzyme mechanisms and the intrinsic electrocatalytic properties and different approaches are possible such as immobilization by adsorption like protein film voltammetry [13,17], or by the use of membranes [18], modified electrodes [19], or in solution [15].…”

Molecular Biology of Microbial Hydrogenases

“…Bruschi et al, 2002) may contribute to the identifi cation of factors indispensable for effi cient intramolecular electron transfer, stability and high turnover of the metallocenter in its supramolecular protein framework. (Cammack et al, 1987;Roberts and Lindahl, 1994;Volbeda et al, 1996;De Lacey et al, 1997), kinetics measurements De Lacey et al, 2000a) and from the crystal structures of oxidized (Volbeda et al, 1995(Volbeda et al, , 1996 and reduced (Garcin et al, 1999;Higuchi et al, 1999) forms of [NiFe]-H 2 ases. The oxidized inactive form of the enzyme yields the Ni u -A state, unready to catalyze H 2 activation (i.e.…”

“…Upon reduction, Ni-B yields the EPR-silent intermediate Ni-S. Ni-S occurs in two states in equilibrium, the Ni r -S (ready) state and the Ni a -S (active) state, supposed to be the protonated form of Ni r -S (De Lacey et al, 1997). Each of the active states, Ni a -S, Ni a -C and Ni a -R have been proposed to be involved in the catalytic cycle (Cammack, 2001;Roberts and Lindahl, 1995;De Lacey et al, 2000a). Only the Ni-C/Ni-R couple is in thermodynamic equilibrium with H 2 .…”

“…Only the Ni-C/Ni-R couple is in thermodynamic equilibrium with H 2 . The competitive inhibitor CO was proposed to block electron and proton transfer at the active site by binding to the Ni atom and thereby displacing H 2 and stabilizing a Ni-S(CO) reduced state De Lacey et al, 2002) (Scheme adapted from De Lacey et al, 2000a;Bleijlevens et al, 2001 andJones et al, 2003). The subscript "u" stands for unready, "r" for ready and "a" for active.…”