Using direct electrochemistry to probe rate limiting events during nitrate reductase turnover
2000
Protein
film voltammetry of the nitrate reductase Paracoccus pantotrophus NarGH reveals a
catalytic response whose shape, magnitude and position reflect intrinsic thermodynamic and
kinetic properties of the enzyme. Under steady state conditions the catalytic wave is displaced to more negative potentials as the substrate concentration is increased and reaches a limiting value of 7.5±10 mV for concentrations exceeding 1 mM nitrate, pH
6. The shape of the wave remains in general agreement with that predicted for an
napp≈1 process across the complete range of nitrate concentrations investigated
and the magnitude of the catalytic response varies in agreement with a Michaelis–Menten description of enzyme kinetics. NarGH is a multi-centred redox enzyme containing
iron–sulfur clusters in addition to a molybdenum–bis-molybdopterin guanine dinucleotide cofactor, Mo(MGD)2, capable 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 chemical
transformations within
the semi-reduced form of the active site.
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