The Hemoprotein Enzymes

The importance of iron enzymes in respiration has been thoroughly established by the work of Warburg, Keilin, and Theorell. Whenever isola- 

The oxidized forms show diffuse bands at 567 and 529 m/i. The a, b, and c bands are the a bands of cytochromes a, b, and c, respectively, while the d band is formed by the fused P bands of cytochromes b and c. The region between 415 and 455 m/A contains the very intense 7, or Soret, bands which are not readily observed with the visual spectroscope. Under some conditions the a band can be shown to be due to two components, one of which forms a complex with carbon monoxide. This has been designated by Keilin and Hartree as cytochrome as . Only one of the cytochromes, cytochrome c, has been obtained in soluble form and purified. The structure and function of the a and b cytochromes is not completely understood. A convenient source for the study of the cytochromes is the heart muscle preparation of Keilin and Hartree which consists of a suspension of fine particles prepared from washed minced heart muscle. 

Isolated cytochrome c in neutral solution shows absorption bands at 408, 530, and 695 m/x in the oxidized form and at 415, 520, and 550 m/x in the reduced form. The heme group is not readily removed by treatment with acid acetone. Following hydrolysis with hydrochloric acid a porphyrin can be isolated which is referred to as porphyrin c. This porphyrin differs from protoporphyrin IX in the nature of side chains 2 and 4 in which the vinyl groups are replaced by thioether bridges with cysteine (Fig. 10). 

Oxidized cytochrome c reacts slowly with cyanide to form a stable com-plexi47,148 which the absorption band at 695 m/x disappears and the band 

Warburg, Heavy Metal Prosthetic Groups, Clarendon Press, Oxford, 1949. 

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Cytochrome P450 (CYP) mono-oxygenases, also called mixed function oxidases, are versatile hemoprotein enzymes that catalyze the cleavage of molecular oxygen to incoiporate one oxygen atom into a substrate molecule and one atom into water [1]. The general stoichiometry of the reaction is as follows (S-H, substrate) ... 

The alteration of hemoprotein(s) P-450 subpopulations in the rat may be observed spectrally, because after treatment of rats with polycyclic aromatic hydrocarbons, the Soret maximum of the carbonmonoxyferrocytochrome complex undergoes a hypsochromic shift from 450 to 448nm (50). This blue shift was not seen with rainbow trout hepatic microsomes (29,30). However, this does not preclude the induction of novel hemoproteins P-450 since (a) the induced hemoprotein(s) maty not differ spectrally from the constitutive enzymes and (b) the induced-hemoprotein may account for only a small proportion of total hemoprotein P-450, and hence its contribution to the position of the Soret maximum of carbon monoxide-treated reduced microsomes may be negligible. The latter suggestion is supported by the work of Bend et al. with the little skate. These workers have shown that hepatic microsomes from 1, 2,3,4-dibenzanthracene treated skates did not exhibit a hypsochromic shift when compared to control microsomes, however, partially purified hemoprotein exhibited an absorbance maxima at 448 nm (51). 

Cytochrome P-450 enzymes have been isolated from a variety of mammalian tissues, insects, plants, yeasts and bacteria. The P-450 cytochromes (Gunter and Turner, 1991) are membrane bound mono-oxygenase enzymes which catalyse oxygen atom transfer to entrapped non-polar substrates. The binding of carbon monoxide to the enzyme produces a split in the 420 nm Soret band to give bands at 364 and 450 nm. The absorption at 450 nm distinguishes the hemoprotein from all others and hence provides... 

Boddupalli, S. S., Hasemann, C. A., Ravichandran, K. G., Lu, J. Y., Goldsmith, E. J., Deisenhofer, J., and Peterson, J. A. 1992a. Crystallization and Preliminary-X-Ray Diffraction Analysis of P450terp and the Hemoprotein Domain of P450bm-3, Enzymes Belonging to 2 Distinct Classes of the Cytochrome P450 Superfamily. Proc. Nat.l Acad. Sci.,89, 5567-5571. 

The toxicological implications in the effect of the respiratory poisons on the enzyme systems of mammals are not fully comprehended, even at this stage of knowledge. For instance, Dixon and Webb (44) point out that the respiration of most animal tissues is insensitive to carbon monoxide which, in the blood, competes with oxygen for the reduced hemoproteins whereas cyanide has a broad inhibitory spectrum which includes various oxidative systems at cellular level and, most importantly, the oxidized forms of the hemoproteins, especially methemoglobin. In this latter connection, phenazine methosulfate has recently been found effective as an experimental therapeutic in cyanide poisoning of mice (13). The respiratory poisons have just been reviewed by Hewitt and Nicholas (72). 

By treatment with 5 M urea and chromatography on DEAE-cellulose, it has been possible to dissociate the E. coli NADPH-sulfite reductase into a flavoprotein and a hemoprotein fraction. The flavoprotein fraction has been shown to be an octamer of a single polypeptide of molecular weight 58,000-60,000 and to contain FMN and FAD in equimolar amounts, but no heme, nonheme iron, or labile sulfide. The hemoprotein fraction is a tetramer of a polypeptide of molecular weight 54,000-67,000, and contains heme, nonheme iron, and labile sulfide, but no flavin. Thus NADPH-sulfite reductase is considered to be an enzyme of agfit subunit composition. The amino acid composition of the whole enzyme and the flavoprotein and hemoprotein fractions have been determined (414). 

S. typhimurium enzyme (S94), which appears to be essentially identical to the E. coli sulfite reductase, are in agreement with the above results. Thus mutants lacking the flavoprotein or the hemoprotein component of the enzyme and containing only the appropriate partial activities have been obtained and the respective partial enzymes isolated. The absorption spectra of sulfite reductase preparations from the wild type and from these mutants are shown in Fig. 46, and the proposed structure for the two components of the wild-type enzyme is shown in Fig. 47. Reconstitution of NADPH-sulfite reductase by recombination of the flavoprotein... 

Protohemin is the major structural component of the active site, and, indeed, is the principal determinant of activity and specificity in hemopro-teins. While hemoproteins are generally categorized into the three classes of oxidases, peroxidases, and electron transport components, the first two groups may have a number of features in common in that the oxidase activity may involve certain steps in which peroxide compounds are involved, and the peroxidases may participate in oxidase activities under appropriate conditions. Nevertheless, it is the purpose of this contribution to explore how the apoenzyme determines the fine structure of the catalytic activities of the hemoprotein 71). In this context, two hypotheses merit consideration (1) that constraints imposed by the protein modulate hemin properties [1, 72-74) nd (2) that the protein participates intimately in the catalytic function(s) of the enzyme 17, 75). 

Fig. 21. Typical intermediates in hemoprotein enzyme active sites. The iron protoporphyrin IX cofactor (heme) forms dioxygen adducts termed oxy-species. In the course of oxygen activation and catalytic redox transformations, the oxy form can be consecutively converted into hydroperoxo- and oxo-type intermediates, which are usually referred to as compound 0, compound I, and compound II. Reproduced with permission from Ref (183). Copyright Nature Publishing Group.

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