Cytochrome bs-reductase

Another pathway is the L-glycerol 3-phosphate shuttle (Figure 11). Cytosolic dihydroxyacetone phosphate is reduced by NADFl to s.n-glycerol 3-phosphate, catalyzed by s,n-glycerol 3-phosphate dehydrogenase, and this is then oxidized by s,n-glycerol 3-phosphate ubiquinone oxidoreductase to dihydroxyacetone phosphate, which is a flavoprotein on the outer surface of the inner membrane. By this route electrons enter the respiratory chain.from cytosolic NADH at the level of complex III. Less well defined is the possibility that cytosolic NADH is oxidized by cytochrome bs reductase in the outer mitochondrial membrane and that electrons are transferred via cytochrome b5 in the endoplasmic reticulum to the respiratory chain at the level of cytochrome c (Fischer et al., 1985). 

Y4. Yubisui, T., Naitoh, Y., Zenno, S., Tamura, M., Takeshita, M., and Sakaki, Y., Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome bs reductase. Proc. Natl. Acad. Sci. U.S.A. 84,3609-3613(1987). 

Recent studies suggest that many factors may affect hydroxyl radical generation by microsomes. Reinke et al. [34] demonstrated that the hydroxyl radical-mediated oxidation of ethanol in rat liver microsomes depended on phosphate or Tris buffer. Cytochrome bs can also participate in the microsomal production of hydroxyl radicals catalyzed by NADH-cytochrome bs reductase [35,36]. Considering the numerous demonstrations of hydroxyl radical formation in microsomes, it becomes obvious that this is not a genuine enzymatic process because it depends on the presence or absence of free iron. Consequently, in vitro experiments in buffers containing iron ions can significantly differ from real biological systems. 

Examination of one real-life case may benefit the reader s understanding. Strittmatter studied the primary kinetic isotope effects arising in the NADH-dependent cytochrome bs reductase (EC 1.6.2.2). The oxidation of NADH and subsequent reduction of cytochrome bs is facilitated by the enzyme-bound FAD group, and the kinetics of the direct transfer of a hydrogen from the A-face (or pro-R) of NADH to the flavin can be monitored by the loss of the 340 run absorbance of the NADH s dihydropyridine ring. Using deuterated isotopic isomers of NADH and several related compounds, Strittmatter obtained the primary kinetic isotope effect data compiled in the table below. 

This FAD-dependent enzyme [EC 1.6.2.2], also known as cytochrome bs reductase, catalyzes the reaction of NADH with two molecules of ferricytochrome bs to produce NAD and two ferrocytochrome bs. 

CYTOCHROME cj HYDROGENASE CYTOCHROME c PEROXIDASE CYTOCHROME P-450 REDUCTASE MIXED-EUNCTION OXIDASE NADH-CYTOCHROME bs REDUCTASE NADH DEHYDROGENASE NITRATE REDUCTASE... 

IMIDAZOLEACETATE HYDROXYLASE NADH-CYTOCHROME bs REDUCTASE NADH DEHYDROGENASE... 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

A mechanism has been proposed for NADH-cytochrome bs reductase based on these elegant studies (Fig. 17) (308, 355). The hydrogen which is stereospeciflcally and directly transferred has been printed in boldface. Catalysis proceeds in a clockwise direction, and after the first catalytic cycle the dissociation of oxidized pyridine nucleotide and the association of reduced pyridine nucleotide are shown as a single step since both are very rapid processes and this emphasizes that the thiol is not (kinetically speaking) exposed during catalysis. The interaction of the thiol and the... 

The mechanism for NADH-cytochrome bs reductase described in Section VII,B was worked out with the soluble enzyme, and the question of its applicability to the interaction of the amphipathic proteins can now be considered. 

Studies on the mechanism of NADPH-cytochrome P-450 reductase have been carried out thus far only with the trypsin- or lipase-solubilized forms. Assuming that this enzyme is composed of several semi-autonomous domains, and assuming further that modification during solubilization is restricted to the domain involved in the interaction with cytochrome P-450, then, as was the case with NADH-cytochrome bs reductase, mechanism studies on the soluble enzyme will contribute to the ultimate understanding of the operation of the reconstituted system. The fact that the soluble reductase is composed of a single polypeptide chain gives hope that the modification is a subtle one. 

FIGURE 16.13 Partial scheme for the metabolism of diethylstilbestrol (DES). DES is administered as the trans isomer (E-DES), which, in solution, is in equilibrium with the cis isomer (Z-DES). Cytochrome P450 enzymes oxidize E-DES and Z-DES to a postulated chemically reactive semiquinone (1), which is further oxidized to a quinone (2), thereby generating reactive oxygen species (ROS) that oxidize cellular macromolecules. Redox cycling is perpetuated and ROS formation is amplified by two enzymes, cytochrome P450 or cytochrome bs reductase, which reduce the quinone back to the semiquinone. The unstable semiquinone and diol epoxide (3) metabolites are presumably those that bind to DNA to form adducts and initiate carcinogenesis. 

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