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 (E'0) (25 degrees 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 E'0 = -174 mV (n = 1) for the haem and E'0 = -3 mV and E'0 = -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.
Kay
C J
CJ
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612.
Barber
M J
MJ
Solomonson
L P
LP
Kau
D
D
Cannons
A C
AC
Hipkin
C R
CR
D002175:Candida / Q000201:enzymology*; D007700:Kinetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D011199:Potentiometry; D013053:Spectrophotometry; D013816:Thermodynamics
D009565:Nitrate Reductases; D050901:Nitrate Reductase