Cytochrome component isolation

Fig. 8. Difference spectra of the three spinach cytochromes isolated by imposing appropriate redox potentials In the sample and reference. See text for details. Figure source DS Bendall, HE Davenport and R Hill (1974) Cytochrome components of the higher plants. Methods in Enzymology 23 341.

The ultimate test, but not one achieved easily, is always the isolation and purification of individual cytochrome components. Cytochrome c has been studied more extensively than b or a cytochromes just because it can be isolated easily, and Table XX is an indication of the success to date. When possible, reconstitution experiments between a purified, extracted component and membrane fragments depleted of that component can provide strong evidence for function. Unfortunately, bacterial systems have proved to be more sensitive to handling then mitochondria, and extraction of cytochrome usually denatures the chain irreversibly (368). 

The first of these new, electron transferring components was coenzyme Q (CoQ). Festenstein in R.A. Morton s laboratory in Liverpool had isolated crude preparations from intestinal mucosa in 1955. Purer material was obtained the next year from rat liver by Morton. The material was lipid soluble, widely distributed, and had the properties of a quinone and so was initially called ubiquinone. Its function was unclear. At the same time Crane, Hatefi and Lester in Wisconsin were trying to identify the substances in the electron transport chain acting between NADH and cytochrome b. Using lipid extractants they isolated a new quininoid coenzyme which showed redox changes in respiration. They called it coenzyme Q (CoQ). CoQ was later shown to be identical to ubiquinone. 

In this text, iron-sulfur clusters are discussed because they appear in proteins and enzymes (1) cytochrome b(6)f, Rieske [2Fe-2S] cluster (Section 7.5 and Figure 7.26) (2) cytochrome bci, Rieske [2Fe-2S] cluster (Section 7.6 and Figure 7.30) and (3) aconitase, [4Fe-4S] cluster (Section 7.9.2.1, and Figure 7.50). The iron-sulfur protein (ISP) component of the cytochrome b(6)f and cytochrome bci complexes, now called the Rieske ISP, was first discovered and isolated by John S. Rieske and co-workers in 1964 (in the cytochrome bci complex). More information about the RISP is found in Section 7.5.1. Section 7.9.2 briefly discusses other proteins with iron-sulfur clusters—rubredoxins, ferrodoxins, and the enzyme nitrogenase. The nitrogenase enzyme was the subject of Chapter 6 in the hrst edition of this text— see especially the first edition s Section 6.3 for a discussion of iron-sulfur clusters. In this second edition, information on iron-sulfur clusters in nitrogenase is found in Section 3.6.4. See Table 3.2 and the descriptive examples discussed in Section 3.6.4. 

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

Because of the difficulty of isolating the electron transport chain from the rest of the mitochondrion, it is easiest to measure ratios of components (Table 18-3). Cytochromes a, a3, b, cv and c vary from a 1 1 to a 3 1 ratio while flavins, ubiquinone, and nonheme iron occur in relatively larger amounts. The much larger... 

The discussion to this point has focused on the isolation of intact mitochondria. By various chemical and physical treatments, mitochondria may be separated into their four components. This allows biochemists to study the biological functions of each component. For example, by measuring enzyme activities in each fraction, one can assign the presence of a particular enzyme to a specific region of the mitochondria. Studies of mitochondrial subfractions have resulted in a distribution analysis of enzyme activities in the four locations (Table E10.1). This type of study is often referred to as an enzyme profile or enzyme activity pattern and the enzyme may be considered a marker enzyme. For example, cytochrome oxidase, which is involved in electron transport, is a marker enzyme for the inner membrane. 

The cytochrome o from Azotobacter vinelandii is reported to consist of one polypeptide of molecular weight 28 000 with two identical heme components. It has also been isolated from the thermophile PS3,1319 Escherichia coli, Vitreoscilla, Pseudomonas aeruginosa, and Rhodopseudomonas spp. The enzyme from Vitreoscilla consists of two identical polypeptides of molecular weight 13 000 and two moles of protoheme IX. A cytochrome b562-o complex from E. coli contains two peptides and, strangely, copper.1320... 

The components of the electron-transfer chain in mitochondria are shown in Figure 69. The sites at which ATP is synthesized are noted. The cytochromes may be characterized by UV-vis studies on membranes, usually by difference spectra. The iron-sulfur proteins may be identified by ESR spectroscopy, but MCD may be of particular value in characterizing isolated clusters. 

Now that it is established that cestodes possess all the components of a electron transport system, is the latter functional Weinbach von Brand (952) failed to demonstrate either respiratory control or oxidative phosphorylation in T. taeniaeformis, although they regarded this as a technical rather than a physiological problem. However, there is good evidence that isolated mitochondria from M. expansa (124-127) and H. diminuta (663, 978) are capable of oxidative phosphorylation and respiratory control. The demonstration that a preparation of H. diminuta mitochondria will oxidise a range of substrates, exhibiting respiratory control, is shown in Table 5.14. Similarly, mitochondria from Diphyllo-bothrium latum can oxidise NADH (728) and succinate (729). It is likely that the classical mammalian-type part of the cytochrome chain in cestodes is capable of oxidative phosphorylation, but there is no evidence for ATP synthesis occurring on the alternative branch from the quinone or vitamin K/cytochrome b complex to cytochrome o. 

As with all the other photosynthetic membrane complexes, the genes for the components of the cytochrome complex are distributed between the nuclear and chlo-roplast genomes of higher plants. The chloroplast genes for the Cyt /, Cyt 6-563 and 17 kDa polypeptides have been extensively characterized, but the nuclear gene(s) for the Rieske Fe-S protein have not yet been isolated. 

The isolation of an active, structurally intact complex was obtained using an association of cholate and octylglucoside and sucrose gradient centrifugation [111]. This preparation did not contain cytochrome 6-559 and possessed a plastoquinol-plastocyanine oxidoreductase activity, inhibited by specific inhibitors (DBMIB, UHDBT). The complex was essentially free of chlorophyll and contaminations by other membrane components, specifically of the ATPase complex. 

Halestrap initially concluded that the increases in mitochondrial pyruvate transport and carboxylation were due to an increase in A pH secondary to stimulation of the electron transport chain in the cytochrome fee, region [255]. The conclusion was based largely on spectral measurements of the redox state of these cytochromes in the control and stimulated states. The spectral measurements were later found to be artifactual due to low amplitude Ca swelling of the mitochondria. Halestrap then suggested that the stable changes in the mitochondria might reside in the lipid components of the membrane due to phospholipase A 2 activity [261,262], but he has been unable to confirm this with lysophospholipid measurements [263]. On the other hand, using an EPR spin label probe of the lipid environment of the isolated mitochondria, Hoek has found differences between control and treated mitochondria [264]. 

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