SPECTROSCOPIC, THERMODYNAMIC AND KINETIC-PROPERTIES OF CANDIDA-NITRATOPHILA NITRATE REDUCTASE

Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H:nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b5-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g1 = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A(1H)(av.) = 1.4 mT]. Oxidation-reduction midpoint potentials (E0') (25°C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of E0' = -174 mV (n=1) for the haem and E0' = -3 mV and E0' = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.