Cytochrome mutagenesis

Szklarz GD, Halpert JR. Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450. Life Sci 1997 61 2507-20. 

Torres, E. Sandoval, J. V. Rosell, F. I., et al., Site-Directed Mutagenesis Improves the Biocatalytic Activity of Iso-l-Cytochrome-C in Polycyclic-Hydrocarbon Oxidation. Enzyme and Microbial Technology, 1995. 17(11) pp. 1014—1020. 

The selective monohydroxylation of heterocyclic compounds such as piperidine derivatives1741 and the 7-lactam (19)[751 have been studied. It is also been shown that hydroxylation of phenylcyclohexane can be effected using cytochrome P450 and the regioselectivity of hydroxylation can be altered by site-directed mutagenesis of the enzyme1761. 

Studies of Cytochrome c Variants Produced by Site-Directed Mutagenesis. 138... 

One example of the combined use of site-directed mutagenesis with chemical modification has been provided by the work of Bowler et al. with yeast iso-l-cytochrome c [15]. These workers introduced a His residue at position 62 to provide a site for attachment of a pentammineruthenium complex. Introduction of a second redox-active metal center to the protein at this position permitted... 

While the results of this work are encouraging, it is clear that the structural definition of mutant proteins of this type is critical to development of rational interpretation of the results if for no other reason than that the structural perturbation introduced is presumably greater than for simple point mutations. Moreover, it would be particularly interesting to compare the functional properties of mutants compared in this manner in assays involving protein-protein reactions relevant to the species of cytochrome c on which the mutagenesis is based. For example, comparison of the activities of wild-type yeast cytochrome c with that of a loop-insertion mutant modelled on a photosynthetic cytochrome c in the reaction with the photosynthetic reaction center could help define the structural elements involved in the cytochrome c binding domain for the reaction center. 

As part of a subsequent study concerning primarily second-site revertant yeast iso-l-cytochrome c variants, Hazzard et al. evaluated the effect of converting Lys-72 to an aspartyl residue by site-directed mutagenesis on the electron transfer kinetics of the cytochrome c-cytochrome c peroxidase complex [136]. Lys-72 was of interest for this purpose, because it is involved in the hypothetical model for the complex formed by these two proteins that was proposed by Poulos and Kraut on the basis of molecular graphics docking [106]. In these... 

Sorrell and co-workers have used site-directed mutagenesis of yeast iso-2-cytochrome c to identify what may be the first functional axial ligand mutant of... 

Prior to the advent of site-directed mutagenesis as a viable technique for the production of specifically modified proteins, the last major event to exert a major influence on the study of protein structure and function was the development of X-ray diffraction analysis for the detailed structural analysis of macromolecules. In the intervening thirty years, the availability of protein structures obtained in this manner combined with a wide range of physical and chemical studies of these proteins allowed development of substantial insight into the relationship between the structure of a protein and its functional attributes. There was some reason to expect, therefore, that functional characterization of specifically mutated proteins based on understanding developed with more classical techniques should permit efficient confirmation of existing hypotheses, particularly for proteins for which the available literature is as extensive as that for cytochrome c. 

Recent structural studies and electron transfer kinetic experiments focus on structures in which a site-specific covalent crosslink between cytochrome c and cytochrome c peroxidase subunits exists. One of these used site-directed mutagenesis to form a disulfide bond between a V197C mutant CcP and an A81C... 

Dichlorobenzidine is an effective inducer of its own metabolic activation (Iba 1987a). The enhancement of 3,3 -dichlorobenzidine mutagenesis has been associated with the induction of cytochrome P-450d (Iba and Thomas 1988), and may result in the elevation of its carcinogenicity. In other animal studies, 3,3 -dichlorobenzidine was also shown to be a potent inducer of hepatic microsomal enzymic activities mediated by cytochrome-P-448 and P-450 (Iba and Sikka 1983 Iba and Thomas 1988). Consequently, it has been suggested that the hepatocarcinogenicity of 3,3 -dichlorobenzidine may be due, at least in part, to the induction of hepatic cytochrome P-488 and DNA-adduction. 

Domanski, T. L. and Halpert, J. R. (2001) Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis. Curr. Drug. M. 2, 117-137. 

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