Studies on NADH(NADPH)-cytochrome c reductase from yeast.
Creators: name: Johnson, Mark Stephen
Publisher: name: University of Utah
Year: 2012
Subjects: Purification
Enzymes

description: An NADH(NADPH)-cytochrome c reductase has been isolated from an acetone powder of a top ale yeast (Saccharomyces cerevisiae, Narragansett) with a purification of 67-fold, a recovery of 2.3% and a final specific activity of 0.406 moles min('-1) mg (U/mg), and 0.312 U/mg for NADH- and NADPH-dependent reduction, respectively.  The enzyme was homogeneous by electrophoresis on cellulose acetate and polyacrylamide gels and had a molecular weight, under non-denaturing conditions (as determined by Sephadex G-100 and Sephacryl S-200 chromatography), of  70,000 daltons.  On denaturation, the enzyme dissociated into two identical subunits which had a molecular weight of 34,000 daltons on sodium lauryl sulfate-urea polyacrylamide gel electrophoresis, an Mw = 35.300 daltons by sedimentation equilibrium in the presence of 4.0 M guanidinium chloride.  Ultraviolet and visible absorbance spectra of the enzyme showed three maxima, at 464 nm, 383 nm, and 278 nm, with the extinction coefficients of 9.88 mM('-1)cm('-1), and 6436 mM ('-1)cm('-1), respectively.  These peaks are typical of flavor-protein, and this reductase contained 0.63 residues of FMN per 34,000 daltons subunit, as evidence by fluorescent analysis under acidic conditions and thin-layer chromatography.  FMN fluorescence was quenched by approximately 96% after association with the reductase.  Metal analysis, by an atomic emission spectrophotometer, revealed no copper, iron molybdenum, or zinc-metals typically associated with flavor-proteins.  Aerobic reduction of the enzyme was possible with dithionite, but not with NAD(P)H.  Anaerobically, both pyridine nucleotides were capable of a two-electron reduction, but without the apparent formation of a spectrophotometrically observable semiquinone intermediate.  Incubation of the apo- or holoenzyme in 1 mM 5,5'-dithiobis(2-nitrobenzoic acid) at pH 7.6 or titration of the holoenzyme with 4,4'-dithiodipyridine at pH 3.05 showed no reactiave thiols, but amino acid analysis revealed the presence of one cysteine (measured as cysteic acid) per subunit.  Sodium lauryl sulfacte polyacrylamide gel electrophoresis in the absence of mercaptoethanol exhibited a single band of identical molecular weight, indicating that a disulfide linkage between subunits was not formed.  Electrophoretic mobility studies on cellulose acetate strips indicated an isoelectric point of pI = 5.25 (ionic strength = 0.05).  Activities measured at various pH values showed an optimum (for both NADH- and NADPH-dependent activity) of pH 7.7.  The reductase was stable over a wide range of pH values.  A kinetic method was used to estimate the dissociation constant for FMN which was 1.53 x 10('-8) M.  This value was similar to the Michaelis constant for FMN (approximately 1.6 x10('-8)M).  A hybrid random-ordered mechanism, where NAD(P)H and FMN add randomly followed by the addition of cytochrome c, was consistent with the kinetic results for both pyridine nucleotides.  In addition, NADPH-dependent cytochrome c reduction was also consistent with kinetics of an ordered mechanism.  The following kinetic values were derived for the reduction of cytochrome c in the presence of 1.73 x 10('-6) M menadione (vitamin K3) and 2.48 x 10('-6) M FMN:  (with NADPH) Km,NADPH = 5.7 (±0.27) x 10('-8) M; Ki,NADPH = (±0.4) x 10('-9) M (for a random hybrid mechanism and 6.7 x (±2.0) x 10('-7) M for an ordered mechanism); Km,cyt c3+ = 1.95 (±0.03) x 10('-6) M; Vmax/Et = 3.8 (±0.1) U/mg = 129.1 min('-1). (with NADH) Km,NADH = 5.9 (±0.05) x 10('-6) M; Ki,NADH = 6.0 (±0.2) x 10('-6) M; Km,cyt c3+ = 1.9 (±0.5) x 10('-6) M; Vmzx/Et = 2.17 (±0.37) U/mg = 73.8 min('-1).  The oxidized forms of the pyridine nucleotides were competitive with their respective reduced forms, and showed inhibition constants of Ki,NADH = 9 x 10('-4) M and Ki,NAD = 1.5 x 10('-2) M.  Arrhenius plots for cytochrome c reduction revealed large differences for the two pyridine nucleotides, with values at 30°C of 6 Kcal/mole and of 11.8 Kcal/mole for dela-Eact for NADH and NADPH, respectively.  Some curvature was evident in the plot of the latter and is likely due to ionic inhibition by the phosphate buffer employed for assay.  Menadione accelerated reductase-mediated reduction of cytochrome c by 8-fold with NADPH and 3.5-fold with NADH as the substrate.  Neither vitamin K1 nor coenzyme Q10 affected the rate.  Electron acceptors other than cytochrome c were studied and compared to NADH-dependent cytochrome c reduction (with NADH-cytochrome c reductase activiaty set at 100) as follows:  (with NADPH) cytochrome c, 77 (+ menadione, 616); ferricyanide, 267 (+ menadione, 267); dichlorophenolindohenol (DCIP), 177 (+ menadione 177); oxygen, 8.8 (+ menadione, 88.6; + menadione catalase, 124); (with NADH) cytochrome c, 100 (+ menadione, 353) ferricyanide, 164 (+ menadione, 49; + menadione and catalase, 88).  Superoxide dismutase inhibited cytochrome c reduction.  However, anaerobic cytochrome c reduction was rapid, indicating that superoxice anion was not a requisite for reduction.  Relative values for anaerobid reduction (with aerobic NADH-cytochrome c reduction set at 100)  were calculated for the following:  (with NADPH) cytochrome c, 81.6 (+ menadione, 677); cytochrome b5, 17.1 (+ menadione, 240); DCIP, 177 (+ menadione, 177); (with NADH) cytochrome c, 118 (+ menadione, 504); cytochrome b5, 11.7 (+ menadione, 186); DCIP, 32.7 (+ menadione, 32.7).  No trashydrogenase activity was observed utilizing thionicotinamide analogs.  These analogs did, however, act as substrates for cytochrome c reduction when reduced by a glucose 6-phosphate dehydrogenase system.  Comparisons were made between this enzyme and others with related activities.  Aside from molecular weight and substrate specificities, "Old Yellow Enzyme" has many similar characteristics.  Difference spectra of crude extracts of this yeast revealed the absence of components of the mitochondrial electron transport chain (the absence of cytochrome oxidase was corroborated enzymatically) and the presence of cytochrome b5 (13.7 nmole/g yeast dry weight).  A large 420 nm peak observed in the reduced (carbon monoxide saturated) minus reduced spectra, together with a shoulder at 488 nm-450 nm, suggested that P-450 was also present.