Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

Abstract

Site-directed mutagenesis of Thr 66 in porcine liver NADH-cytochrome b 5 reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k cat values of this mutant were ϳ10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k red ) was consistent with the k cat values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the ratelimiting step. In the T66A mutant, the k red value was larger than the k cat values, but the k red value in the presence of NAD ؉ was consistent with the k cat values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD ؉ . These data suggest that the oxidized T66A-NADH-NAD ؉ ternary complex is a major intermediate in the turnover and that the release of NAD ؉ from this complex is the rate-limiting step. These results substantiate the important role of Thr 66 in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochrome b 5 .NADH-cytochrome b 5 reductase (EC 1.6.2.2) is a member of the large family of flavin-dependent oxidoreductases that transfer an electron from two-electron carriers of nicotinamide dinucleotides to one-electron carriers such as heme proteins and ferredoxins. This enzyme catalyzes the electron transfer from NADH to cytochrome b 5 (b5) 1 (1-3), and participates in fatty acid synthesis (4, 5), cholesterol synthesis (6), and xenobiotic oxidation (7) as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, this enzyme participates in the reduction of methemoglobin (8).The outline of the catalytic cycle of the solubilized catalytic domain of NADH-cytochrome b 5 reductase (b5R) is understood as follows (3) (Scheme I). At first, two electrons are transferred from NADH to FAD by hydride (H Ϫ ) transfer. Then the twoelectron reduced enzyme-NAD ϩ complex (E-FADH Ϫ -NAD ϩ ) transfers two electrons to two one-electron acceptors one by one via the anionic red semiquinone form (E-FAD⅐ Ϫ -NAD ϩ ), and the reduced enzyme returns to the oxidized state. Strittmatter (9 -11) suggested that the reduction of FAD by NADH is the rate-limiting step in electron transfer catalyzed by b5R. Iyanagi et al. (3,12) found that the anionic red sem...

The Role of a Conserved Serine Residue within Hydrogen Bonding Distance of FAD in Redox Properties and the Modulation of Catalysis by Ca2+/Calmodulin of Constitutive Nitric-oxide Synthases

“…In the case of FNR, this conserved serine residue is primarily involved in hydride ion transfer by affecting the interaction between the nicotinamide ring of NADP ϩ and FAD (39). In cytochrome b 5 reductase, the threonine residue at this position plays an important role in catalysis and electron transfer (40). The intricate hydrogen bonding network of this serine residue, along with conserved cysteine, aspartate, and tryptophan residues in CYPOR (16), has been implicated in hydride ion transfer in CYPOR (41).…”

The molybdenum cofactor enzyme mARC: Moonlighting or promiscuous enzyme?

“…Cytb5 and Cytb5‐R are important electron transport proteins involved in many different physiological processes. Cytb5‐R is a flavoenzyme that is able to transfer electrons from NADH to the heme‐containing Cytb5 . However, in contrast to human mARCs, E. coli YcbX contains a Ferredoxin (Fd) [Fe2‐S2] domain fused to the C‐terminus domain that is essential for its activity (Fig.…”

NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family

“…In addition to the characteristic absorbance peak at 596 nm, however, the semiquinone state of FAD shows characteristic peak with absorbance at ~520 nm [94–98]. This peak is also observed in neutral semiquinones in FADH of spinach FNR [99], and the Thr66Val mutant of NADH-cyt b5 reductase [100]. This peak is also observed as a ~520 – 530 nm shoulder during the reduction of oxidized nNOSred by NADPH in the presence of CaM (see Fig.…”