Oxidation-reduction mechanisms, cytochrome

Table IV also lists the structure analyses of three bacterial cytochromes. Logically these should be delayed to the second half of the chapter, but the results have had such an important influence on thinking about oxidation-reduction mechanisms that their discussion here is mandatory. A key structure study from an evolutionary standpoint was that of R. rvhrum by Salemme, Kraut, and colleagues at the University of California, San Diego (30,31). That study established in one stroke that the eukaryotic cytochrome fold also extended to a bacterial cytochrome and included the cytochromes of photosynthesis as well as respiration. The fact that this structural homology had been predicted on the basis of amino acid sequence comparisons (32) did not lessen the excitement of seeing direct confirmation from the molecular model. Only one of three possible conclusions can be drawn ...

Recent chemical evidence has made it apparent that both of the two detailed oxidation-reduction mechanisms which have been proposed for cytochrome must be abandoned the Winfield mechanism espoused for eukaryotic c, and the heme crevice hydrogen bond network proposed for bacterial c-i. Each mechanism has been undermined by the discovery of unworkable amino acids at key positions in species for which the proposed scheme should hold. A fresh approach is needed. 

Shiro, Y., M. Fujii, T. lizuka, S.I. Adachi, K. Tsukamoto, K. Nakahara, and H. Shoun (1995). Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. J. Biol. Chem. 270, 1617-1623. 

Phase I metabolic reactions involve oxidation, reduction, or hydrolysis of the parent molecule, resulting in the formation of a more polar compound. Phase 1 reactions are mediated by the cytochrome P450 (GYP) family of enzymes. While metabolism used to be thought of as the body s detoxification process, phase I metabolites may be equally or even more pharmacologically active than the parent compound. Drug metabolism in general, and CYP-based mechanisms in particular, are discussed in detail in Chapter 5. 

Most of the numerous other riboflavin-containing enzymes contain FAD. FAD is an integral part of the biological oxidation-reduction system where it mediates the transfer of hydrogen ions from NAD11 to the oxidized cytochrome system. FAD can also accept hydrogen ions directly from a metabolite and transfer them to either NAD, a metal ion, a heme derivative, or molecular oxygen. The various mechanisms of action of FAD are probably due to differences in protein apoenzymes to which it is bound. 

Flavocytochrome b2 catalyses the oxidation of lactate to pyruvate at the expense of cytochrome C. After reduction of flavin (FMN) by the substrate, reducing equivalents are transferred to heme b2 and from there to cytochrome C602. The mechanism of this process has been studied603 at 5.0 °C by determining the D KIE in the FMN reduction using L-[2-2H]lactate and wild-type enzyme and also with the Y143F mutant prepared from transformed Escherichia coli604. Tritium IE in the conversion of [2-3-H]lactate to... 

The second proton transfer mechanism involves protonation of carboxyl or histidyl groups associated with electron carriers in the membrane and release of protons from these sites through proposed channels when the electron carrier is oxidized. This is essentially a proton channel system with movement through the channel gated by the oxidation-reduction state of the prosthetic group on the electron transport protein. The classical example of this is seen in cytochrome c oxidase (Figure 3). 

Cytochrome c, a small heme protein (mol wt 12,400) is an important member of the mitochondrial respiratory chain. In this chain it assists in the transport of electrons from organic substrates to oxygen. In the course of this electron transport the iron atom of the cytochrome is alternately oxidized and reduced. Oxidation-reduction reactions are thus intimately related to the function of cytochrome c, and its electron transfer reactions have therefore been extensively studied. The reagents used to probe its redox activity range from hydrated electrons (I, 2, 3) and hydrogen atoms (4) to the complicated oxidase (5, 6, 7, 8) and reductase (9, 10, 11) systems. This chapter is concerned with the reactions of cytochrome c with transition metal complexes and metalloproteins and with the electron transfer mechanisms implicated by these studies. 

Fig. 5. Comparison of kinetics of cytochrome oxidation and reduction in an anaerobic suspension of intact ceils of the photosynthetic bacterium Chromatium at 300,250 and 77 K. Scaies for the absorbance-change and time as well as the calculated rates of cytochrome oxidation and re-reduction are shown. Figure source left panel from Chance and Nishimura (1960) On the mechanism of chiorophyil-cytochrome interaction The temperature insensitivity of tight-induced cytochrome oxidation in Chromatium. Proc Nat Acad Sci, USA. 46 20 and right panei from Chance and DeVault (1964) On the kinetics and quantum efficiency of the chiorophyil-cytochrome reaction. Ber Bunsenges Phys Chem 68 725.

Before we try to understand the mechanism of oxidative phosphorylation, let s first look at the molecules that carry out this complex process. Embedded within the mitochondrial inner membrane are electron transport systems. These are made up of a series of electron carriers, including coenzymes and cytochromes. All these molecules are located within the membrane in an arrangement that allows them to pass electrons from one to the next. This array of electron carriers is called the respiratory electron transport system (Figure 22.7). As you would expect in such sequential oxidation-reduction reactions, the electrons lose some energy with each transfer. Some of this energy is used to make ATP. 

Production of superoxide radical anion (02 ) during oxidation of dihydroorotate in rat hver mitochondria was not affected by antimycin A, thenoyl-trifluoroacetone, or added ubiquinone but was inhibited by orotate, a product inhibitor of dihydroorotate dehydrogenase (Forman and Kennedy 1975). It appears likely that superoxide is generated at the primary dehydrogenase. Dihydroorotate dehydrogenase differs from succinate dehydrogenase both in its utilisation of ubiquinone and in the mechanism of cytochrome b reduction. Formation of orotate is only partially inhibited by thenoyltri-fluoroacetone and the inhibitor does not prevent the reduction of cytochrome by dihydroorotate. 

The sequence of reactions in which the cytochromes participate is a mechanism for transferring electrons to molecular oxygen via iron complexes that are alternately in ferric and ferrous states. The order of the transfer has been deduced from studies with inhibitors, in which the electron-transport chain is broken so that components below the break are reduced, those above are oxidized from studies with poised potentials, in which the relative degrees of oxidation and reduction define the oxidation-reduction potentials of the various components and from rapid kinetic measurements, in which the order of reduction or oxidation can be seen. These methods agree on the following sequence ... 

Many investigators have shown that nonspecific reagents as diverse as calcium phosphate gel, EDTA, histidine, and nonspecific proteins activate succinoxidase preparations in otherwise unfavorable environments. The mechanism of the activation is not established, but it has been repeatedly suggested that the activators in some manner influence the steric orientation of components of the particulate succinoxidase. Another component of electron-transport systems has been implicated by Nason and Lehman. DPNH oxidation by a particulate fraction of rat muscle was found to be decreased by extraction of 10 per cent of the lipid with isooctane the activity was restored by addition of a-tocopherol (vitamin E) or the lipid extracted from muscle or bovine serum albumin. These lipids are able to reverse the inhibition of cytochrome c reduction caused by antimycin A. It has not been determined whether the tocoph-... 

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