Cytochrome peroxide-dependent oxidations

The oxidation of cyclohexene by a fully reconstituted cytochrome P-450lm system (5) gave only cyclohexene oxide (1) and cyclohexenol (2) in a ratio of 0.92 1. Results for the peroxide-dependent oxidation of cyclohexene in the presence of cytochrome P 450 are presented in Table I. Inspection of the data suggests that subtle differences exist between the oxidants generated from these four oxidants and lead to the observed sixfold change in the ratio of 1 and 2 in the product mixture. Also apparent is the fact that no obvious correlation exists between the ratio of the products and the nature or effectiveness of the oxidant. 

Peroxide-Dependent Oxidations with Cytochrome P 450. Peroxide-dependent oxidations were carried out exactly as described above except that NADPH-cytochrome P-450 reductase and NADPH were omitted. The peroxide solution (50 fxL of a 20mM solution) was added to the premixed enzyme-substrate solution. The quenching solution was 100 fxL of 30% sodium hydroxide saturated in sodium dithionite. 

Thus, superoxide itself is obviously too inert to be a direct initiator of lipid peroxidation. However, it may be converted into some reactive species in superoxide-dependent oxidative processes. It has been suggested that superoxide can initiate lipid peroxidation by reducing ferric into ferrous iron, which is able to catalyze the formation of free hydroxyl radicals via the Fenton reaction. The possibility of hydroxyl-initiated lipid peroxidation was considered in earlier studies. For example, Lai and Piette [8] identified hydroxyl radicals in NADPH-dependent microsomal lipid peroxidation by EPR spectroscopy using the spin-trapping agents DMPO and phenyl-tcrt-butylnitrone. They proposed that hydroxyl radicals are generated by the Fenton reaction between ferrous ions and hydrogen peroxide formed by the dismutation of superoxide. Later on, the formation of hydroxyl radicals was shown in the oxidation of NADPH catalyzed by microsomal NADPH-cytochrome P-450 reductase [9,10]. 

Thus, the isotope effect for the allylic oxidation of cyclohexene by cytochrome P 450 is about 5 and is the same for the reconstituted, NADPH-dependent and the peroxide-dependent paths. This similarity suggests that although product ratios may change from one oxygen donor to another, the mechanism of oxygen transfer may be invariant. Efforts to develop a clearer understanding of the relationships between the 02-dependent and peroxide-dependent pathways for oxygen transfer catalyzed by cytochrome P 450 are currently underway. 

As important as calcium is probably iron [122]. Iron is the metal center of many essential proteins and enzymes, such as hemoglobin, an oxygen carrier, or peroxidase, that oxidizes hydrogen peroxide, or even the large family of cytochromes, which act as electron transfer proteins in many important biochemical processes [85]. New families of MRI contrast agents have been designed such that their relaxivity is iron concentration dependent [128-130]. The two latest are based on Gd(III) chelates (Fig. 20) but differ by the mechanism responsible for their iron sensitivity and will be described further. 

The role of these interesting plasma membrane-dependent, vanadate-stimulated NAD(P)H oxidation reactions in cellular metabolism remains to be elucidated, although multiple interactions with cellular metabolism and components are possible including interactions with xanthine oxidase and lipid peroxidation [24], Decavanadate has been shown to enhance cytochrome c reduction [31], and cytochrome c release from mitochondria is associated with initiation of apoptosis. Perhaps the reduced cytochrome c is more readily released from the mitochondria. With increasing emphasis on the redox properties of vanadium being important in its pharmacological effects, it is quite possible that these reactions, either protein dependent or not, may play a role in therapeutic actions of vanadium. 

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