Using direct electrochemistry to probe rate limiting events during nitratereductase turnover

Protein film voltammetry of the nitrate reductase Paracoccus pantotrophus N arGH reveals a catalytic response whose shape, magnitude and position refle ct intrinsic thermodynamic and kinetic properties of the enzyme. Under stea dy state conditions the catalytic wave is displaced to more negative potent ials as the substrate concentration is increased and reaches a limiting val ue of 7.5 +/- 10 mV for concentrations exceeding 1 mM nitrate, pH 6. The sh ape of the wave remains in general agreement with that predicted for an n(a pp)approximate to1 process across the complete range of nitrate concentrati ons investigated and the magnitude of the catalytic response varies in agre ement with a Michaelis-Menten description of enzyme kinetics. NarGH is a mu lti-centred redox enzyme containing iron-sulfur clusters in addition to a m olybdenum-bis-molybdopterin guanine dinucleotide cofactor, Mo(MGD)(2), capa ble of accommodating multiple electrons at the enzyme's active site. It is suggested that progression from the semi-reduced to fully-reduced state of the active site, constitutes a rate defining event in the catalytic cycle. Formation of the fully-reduced active site may be limited either by relayed , intramolecular electron delivery to the active site and/or the rates of c hemical transformations within the semi-reduced form of the active site.