NADH-Cytochrome Reductase

Shirabe, K., Yubisui, T. Takeshita, M. (1989). Expression of human erythrocyte NADH-cytochrome 65 reductase as a thrombin-cleavable fused protein in Escherichia coli. Biochimica et Biophysica Acta 1008, 189-92. 

Strittmatter, P., Kittler, J.M., Coghill, J.E. Ozols, J. (1992). Characterization of lysyl residues of NADH-cytochrome 65 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome 65 by site-directed mutagenesis of an expression vector for the flavoprotein. Journal of Biological Chemistry 267, 2519-23. 

Yubisui, T., Shirabe, K., Takeshita, M., Kobayashi, Y., Fukumaki, Y., Sakaki, Y. Takano, T. (1991). Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome 65 reductase. Journal of Biological Chemistry 266, 66-70. 

Lipoamide dehydrogenase (15, 16) and NADH-cytochrome 65 reductase (17) were covered in detail in the second edition of The Enzymes. The material in these chapters has stood the test of time remarkably well and will only need to be summarized here. 

Studies leading to the proposal of a mechanism for NADH-cytochrome 65 reductase were carried out with the snake venom-extracted enzyme,... 

The reactivity of the lysyl residues in NADH-cytochrome 65 reductase has been investigated and found to fall into three groups. Modification of the first most reactive group (about half the total) results in loss of the ability to interact with cytochrome bg. In the presence of NADH all but one of the remaining lysyl residues react, resulting in destabilization of the holoenzyme structure (349). 

Elucidation of the total primary sequence of NADH-cytochrome 65 reductase is now well advanced 347). 

It can be seen that this sequence of reactions is simply an expression in kinetic terms of the mechanism shown in Tig. 17 which was derived for the soluble enzyme. Whereas in the soluble system fca was the rate limiting step, kr (and perhaps fcs) is rate limiting in the bound system. The bound system is therefore diffusion-limited. The data indicate that in the intact microsome, NADH-cytochrome 65 reductase is turning over at about 60% of the maximum velocity sp). 

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 65 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 32.2 Catalytic cycle of cytochrome P450 associated with monooxygenase reactions. [Fe ] = ferricytochrome P450 hs = high spin Is = low spin [Fe ] = ferrocytochrome P450 Fpi = flavoprotein 1 = NADPH-cytochrome P450 reductase Fp2 = NADH-cytochrome 65 reductase cyt 65 = cytochrome 65 XH = substrate (modified from ). 

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