Cytochrome reaction with

FIG. 21 Schematic diagram of cytochrome P450 mono-oxygenase reaction with substrate RH and product ROH. 

Cytochrome c is a heme containing protein which occurs in muscle at lower concentrations than does myoglobin. It was demonstrated some time ago (18) that oxidized cytochrome c reacts with gaseous nitrite oxide to produce a nltrosyl compound. Recent work (19, 20, 21) has examined the reactions of cytochrome c with nitrite and the contribution of the product formed to cured meat color in considerably more detail. The general conclusion is that even at the pH normally encountered in meat, the reaction can take place in the presence of ascorbic acid but probably does not affect meat color because of the unstable nature of the reaction product and the low concentration. 

Organic peroxides such as cumene hydroperoxide and t-butyl hydroperoxide have extensively been used as experimental agents. They provoke lipid peroxidation in hepatocytes, probably by the generation of alkoxyl and peroxyl radical intermediates after reaction with cytochrome P450. Other cytotoxic mechanisms are probably involved including protein thiol and non-protein thiol oxidation and deranged calcium homeostasis (Jewell et al., 1986). In fact, the addition of cumene hydroperoxide to isolated bUe duct cells, devoid of cytochrome P450 activity, still results in cell death but lipid peroxidation is not detectable (Parola et al., 1990). 

Wever R, Van Gelder BF, Der Vartanian DV. 1975. Biochemical and biophysical studies on cytochrome c oxidase XX. Reaction with sulphide. Biochem Biophys Acta 387 189-193. 

Other redox partners Co(bipy)33+ (oxidant) and Ru(NH3)s py2+ (reductant) are likewise partially blocked by Pt(NH3)6 +. Interestingly the reaction of cytochrome c(II) with PCu(II) is also blocked by Pt(NH3)5 +, thus identifying this as a site for electron transfer with cytochrome c. This observation is con-sis tant with a preliminary report of NMR results (19). The blocking is in fact more extensive than that observed with the above complexes, which is reasonable in view of the larger size of cytochrome c. Reaction with the negatively charged dipicol-inate oxidant, Co(dipic)2, was similarly investigated, where separate association of the oxidant with Pt(NH3)6 + can be... 

Reaction with Chemically Modified Cytochrome c. Chemically modified (CDNP) cytochrome c derivatives have been prepared by Margoliash and colleagues (22). Lysine residues react as in (17),... 

Figure 10. View of cytochrome c with positions of exposed heme edge (block) and lysine modifications (sequence numbers for a-carbons) shown. The smaller circles indicate the relative effectiveness of modifications on rate constants for the reaction of Cyt c(II) + PCu(II) ( see Table III). Preferential interaction with PCu(II) in the direction 25,27,13,87 is indicated.

Such reaction with aromatic hydroperoxides has been studied by Coon and coworkers [54], Instead of hydroperoxides, iodosobenzenes, and iodobezene acetates may participate in Reaction (8) as the oxygen donors [55]. These authors proposed that the reaction of cytochrome P-450 with iodosobenzene proceeds to form an iron-oxo intermediate complex containing only one oxygen atom derived from the substrate. 

It has earlier been suggested to make cytochrome c a more specific reagent for superoxide detection by its acetylation or succinoylation [9-11], It was proposed that acetylation and succinoylation must cause a greater decrease in the reaction of cytochrome c with NADPH cytochrome P-450 reductase than with superoxide due to a decrease in the electrostatic charge of native cytochrome c [12]. However, the rate constant for the most selective succinoylated cytochrome c became about 10% of native cytochrome [13], making this assay even less sensitive. 

Compared with Molsidomine, both SIN-1 and SIN-1A are reported to induce similar, but more rapid hypotensive action [95, 96]. SIN-1A, after undergoing oxidation in the presence of oxygen or, in vivo, possibly by redox-active enzymes such as cytochrome C [97, 98] or by reaction with ferric myoglobin formed during reperfusion injury [99], releases NO through an intermediate radical cation. 

In order to obtain further information on the magnitude of the overall reaction volume and the location of the transition state along the reaction coordinate, a series of intermolecular electron-transfer reactions of cytochrome c with pentaammineruthenium complexes were studied, where the sixth ligand on the ruthenium complex was selected in such a way that the overall driving force was low enough so that the reaction kinetics could be studied in both directions (153, 154). The selected substituents were isonicotinamide (isn), 4-ethylpyr-idine (etpy), pyridine (py), and 3,5-lutidine (lut). The overall reaction can be formulated as... 

Mansuy D, Valadon P, Erdelmeier I, et al. Thiophene S-oxides as new reactive metabolites formation by cytochrome P450 dependent oxidation and reaction with nucleophiles. J Am Chem Soc 1991 113(20) 7825—7826. 

By 1960 it seemed likely that vesicles (sealed membrane preparations) were essential for ATP formation to occur. R.J.P. Williams (1959-1961 see 1993) postulated that complex assemblies of catalysts, as in the cytochrome chain, would allow spatial separation of reaction products within the mitochondrial membrane. The initial site of reaction with... 

Cleavage of the oxirane C-0 bond produces a zwitterionic intermediate (Fig. 10.22), which that can undergo chloride shift (Pathway a) to 2,2-dich-loroacetyl chloride (10.90) followed by hydrolysis to 2,2-dichloroacetic acid (10.91). Furthermore, the zwitterionic intermediate reacts with H20 or H30+ (Pathway b) by pH-independent or a H30+-dependent hydrolysis, respectively. The pH-independent pathway only is shown in Fig. 10.22, Pathway b, but the mechanism of the H30+-dependent hydrolysis is comparable. Hydration and loss of Cl, thus, leads to glyoxylyl chloride (10.92), a reactive acyl chloride that is detoxified by H20 to glyoxylic acid (10.93), breaks down to formic acid and carbon monoxide, or reacts with lysine residues to form adducts with proteins and cytochrome P450 [157], There is also evidence for reaction with phosphatidylethanolamine in the membrane. 

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. 

Rate constants for the reaction of cytochrome f with inorganic oxidants are independent of pH when it is between 6.5-8.0 [150]. At pH < 6.5 protonation does have an effect, and with for example [Co(phen)3] there is a decrease and [Fe(CN)g] an increase in rate constants. From the kinetics cytochrome f shows no association with positively charged [Co(phen)j], and no competitive inhibition is observed with [Pt(NH3)6]" or [(NH3)5CoNH2Co(NH3)5] " [150]. It is possible therefore to study the effect of these reagents on the reaction of cytochrome f(II) with PCu(II), which... 

Studies on the reaction ofP. pantotrophus cytochrome cd with oxygen required that the enz5une be initially in the reduced state. Although the latter can be obtained using dithionite as reductant, this is best avoided... 

The biphasic reaction with CO points to the existence of multiple heme-hemopexin conformers, and this is borne out by spectral analyses. The absorbance spectra of rabbit ferri-, ferro-, and CO-ferro-mesoheme-hemopexin are entirely analogous to those of other bis-histidyl heme proteins such as cytochrome 65 142), but the CD spectra exhibit unusual features (Fig. 11). Of particular interest are the weak signal of the ferro complex and the bisignate signal of the CO-ferro complex (also seen in the NO-ferro-mesoheme-hemopexin complex (140) and in human ferri-protoheme—hemopexin (139)). 

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