Two Haems

Cytochrome Cj contains a c-type haem as prosthetic group in its wedge-shaped N-terminal domain located in the inter-membrane space. This extrinsic domain is anchored to membrane by a transmembrane helix at the C-terminal end (residues 204 to 222 in bovine bc ). This helix runs alongside cytochrome b and can be removed by mild protease treatment or gene truncation to produce a protein fragment (Hase et al., 1987 Li et al., 1981). 

FIGURE 7. Structure of the inter-membrane (external surface) domains of the bc complex viewed from within the membrane, with the transmembrane helices truncated at roughly the membrane surface. Cytochrome Cl and Rieske protein are drawn as cylinders, subunit 7,8,10, and 11 as ribbons. The Ci haem, Rieske Fe2 S 2 cluster and the two disulfide cysteines of subunit 8 are drawn as ball-and-stick models. Cytochrome c 1 is painted in dark gray, the Rieske protein in light gray. 

Alternative Conformations of Rieske Protein and Cross-transfer of Electrons 

It was unexpected that the extrinsic domain of Rieske protein was found at different positions in different crystal forms. In bovine structure IQCR (see Table 2), the location of the Rieske extrinsic domain was identified to be close to cytochrome b (Xia et al., 1997). This domain was suggested to have high mobility based on further inhibitor binding experiments and anomalous scattering studies (Kim et al., 1998). In the native chicken bc structure (IBCC), the Rieske domain was found to be far away from haem Ll and close to cytochrome c. However, in the chicken bci complex co-crystallized with Qo site inhibitor stigmatellin (structure 2BCC and 3BCC) the Rieske domain was close to haem Ll and far away from cytochrome Cl, which is approximately the position found in the structure 

FIGURE 9. Plot of Fej Sj -haem c i and Fez Sa-haem distances for different structures of foci complex. (Adapted from Iwata eta/., 1998) 

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Dus, K., R. C. Bartsch, and M. D. Kamen Amino acid sequence of a haem peptide with two haem groups. Journ. Biol. Chem. 236, PC47 bis PC48 (1961). 

Newton, N. (1969). The two-haem nitrite reductase of Micrococcus denitrificans. Biochitn. Biophys. Acta 185, 316-331. 

Cytochrome c and Cytochrome c Oxidase. - The mitochondrial electron transport chain is the site at which most of the free energy to be obtained from the oxidation of substrates is released and conserved as the energy-rich molecule ATP. In the final stage of this process, CcO, which is supplied with electrons by cyt c, catalyses the four-electron reduction of oxygen to water. Both are haem proteins, with CcO containing two haem and three copper centres, and both exhibit peroxidase-type activity. 

Type 2 centres include superoxide dismutase, discussed above, and cytochrome c oxidase (COX). COX consists of two haem iron centres, cytochrome a and cytochrome a3, and two electronically distinct copper centres, Cua and Cub, which catalyse ... 

The knowledge of these unique short iron-oxygen distances in ferryl compounds aids in identifying whether a compound has a ferryl structure. Thus the 580 nm compound formed upon addition of peroxide to mitochondrial cytochrome c oxidase has an iron-to-oxygen distance of 1.7 A, suggesting that it is a ferryl intermediate [117]. Of the two haem iron atoms in this molecule, one is unreactive to peroxide. Therefore it is possible to analyse an EXAFS spectrum of the peroxide-treated enzyme minus the spectrum of the untreated enzyme to determine this distance. However, clearly such difference EXAFS spectra will have increased errors associated with the estimate of iron-oxygen distances. 

The data given is collected from Refs. 74-79. Cytochrome aa, contains two haems and two coppers per monomer. Considerable variations in the literature are due in part to differences in protein determination, and in part to use of erroneous extinction coefficients. The ratio of Complex 1 Complex 111 Cytochrome c Complex IV Ubiquinone is about 1 4 8 8 64 in most mitochondria. The content of cytochrome c is somewhat variable, however, and is lowered by extensive washing of mitochondria is salt solutions. 

Cytochrome oxidase contains two haem groups and two protein-bound coppers per minimal functional unit, i.e., the monomer (Table 3.4). Apparently a single copy of most polypeptides is present in this monomer. The haems are chemically identical (haem A), but are bound quite differently to the protein, which gives them widely different functions and spectroscopic properties (Table 3.4). The same is true of the two coppers, Cu and Cug. The haems will be called a and a, respectively. As discussed below, it is not certain whether they have different apoprotein parts, i.e., whether they are formally different cytochromes . 

Of the nine other conserved histidines in subunit I, two may be potential ligands of haem a (in case this haem is not associated with subunit II). In such a case subunit I would be similar to cytochrome b of the 6c, complex, which binds two haems (see below). Note that the conserved membranous histidines of subunit I (Fig. 3.5) are all predicted to be located in the lower aspect of the membrane. 

In most potentiometric titrations only two cytochrome b species are distinguished [208-210], i.e., 6-562 and 6-566, with values of about -(-40 and —40 mV, respectively (both pH dependent). However, proposals have been made of up to four different species (see Refs. 202, 208, 211), i.e., two components 6-562 and separate identity of 6-558 and 6-566. Although it now seems clear that there are two haems 6 per monomer, a functional dimer [211] or different functional states of the monomer... 

Electron transfer per se from haem a to the binuclear site, over a distance of ca. 7-9 A between the edges of the two haem groups, is expected to be very fast (t = a few nanoseconds) . Although haem-haem electron transfer with a time constant of ca. 3 ps has been measured in several laboratories, there are indications of a much faster rate of electron tunneling (but see also ref. 30). Yet, the electron transfer rate measured is already about three orders of magnitude faster than E ax, and can hence hardly be the cause of the limitation in the resting enzyme. However, the observed 3 ps electron transfer is an electron equilibration between haems r/j and a after reduction of haem as in the presence of CO, followed by flash photolysis of CO. 

The complex catalyzes electron transfer from reduced UQ to cytochrome c, coupled to the translocation of protons by a mechanism known as the Q cycle [55-57]. This involves the diversion of half of the electrons available from ubiqui-nol oxidation and deprotonation at a site on the outside of the inner mitochondrial membrane (Qo site) to reduce and protonate UQ at a site on the inside of the membrane (Qi site). The pathway for electron transfer across the membrane is provided by the two haem centers (bt and bn) of the mitochondrial gene product cytochrome b. The remainder of the electrons from ubiquinol oxidation pass along the chain to reduce first the Rieske iron sulfur protein (ISP), then cytochrome Cl and then cytochrome c (Fig. 13.1.3). 

Cytochrome c oxidase for higher organisms contains two haem chromophores at different reduction potentials, a and a, each with an associated copper centre, Cua and Cub respectively. The sequence of reduction of molecular oxygen by fully reduced, membrane-bound cytochrome c oxidase has been deduced > using dual-wavelength multichannel spectroscopy at low temperature. Initial binding of O a is followed by formation of superoxide and oxidation of haem a. Subsequent reactions include internal electron transfer from haem a and Cub with formation of peroxide. Ferricyanide pretreatment oxidizes only haem a and its associated copper and the initial interaction with O2 again produces superoxide and oxidized haem a. ... 

Initial studies have shown the importance of the two haem o>ygenase enzymes. A study by Gronert and co-workers compares injury repair rates in mice. HO-2 wild type and knockout mice were wounded and the cornea was stained with fluorescein the rates and quality of healing was then compared. Figure 1 shows the results from this study. 

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