Cytochrome binding sites

Fig. IB shows that in all preparations an excess RC s exists having their cytochrome binding site oriented towards the exterior of the vesicles, particularly in vesicles prepared from LDAO-depleted RC s. However, a complete unidirectional orientation is not obtained under these conditions (contrast Pachence et al., 1979). This becomes more evident when one realizes that at the two lowest lipid/RC ratio s the amount of RC s that are not incorporated into a bilayer, increases significantly. Under these conditions maximally a ratio of 70 to 30 of RC s with opposite orientation occurs. A higher net-orientation can be obtained by lowering the salt concentration.

Upon fractionation of vesicles reconstituted from LDAO-depleted RC s at a molar lipid/RC ratio of 560 on Biogel A-50 it turns out that the sizes of the vesicles vary from 1200 to 500 nm (as measured with light scattering). In these fractions the net-orientation of the RC s decreases with vesicle size from 80/20 to 60/40. This suggests that the RC s spontaneously orient themselves with their cytochrome binding site on the outside of large vesicles, whereas an increased curvature of the bilayer leads to an increased scrambling of the RC s, possibly due to increased steric repulsion. 

In the ci positional state, fast electron transfer from the Rieske protein to cytochrome Ci will he facilitated hy the close interaction and by the hydrogen bond between His 161 of the Rieske protein and a propionate group of heme Ci, but the Rieske cluster is far away from the quinone binding site. 

Hydroquinone will bind in a quinone binding site that is provided by cytochrome b. Before hydroquinone can be oxidized, it must first be deprotonated (step 2). 

Rao, S. B. K., A. M. Tyryshkin et al. (2000). Inhibitory copper binding site on the spinach cytochrome bf complex Implications for Q0 site catalysis. Biochemistry 39 3285-3296. 

On the Model of the Substrate Binding Site of Cytochrome P-450c... 

The finding that BaP is highly stereoselectively metabolized to dihydrodiols and bay-region 7,8-dihydrodiol-9,10-epoxides (summarized in Figure 1) by liver microsomes from 3-methylcholanthrene-treated rats led Jerina et a . (48) to propose a model of the substrate binding site for cytochrome P-450c (Figure 7) which... 

Figure 7. Steric model proposed by Jerina, et al. for the catalytic binding site of cytochrome P-450c (P-448) to account for the stereoselective metabolism of polycyclic aromatic hydrocarbons (48). The boundary should be enlarged in the directions shown to accommodate substrates whose mechanism of stereoselective oxygenation does not fit the steric model originally proposed.

Dabrowski, M.J., Schrag, M.L., Wienkers, L.C. and Atkins, W.M. (2002) Pyrene-pyrene complexes at the active site of cytochrome P450 3 A4 evidence for a multiple substrate binding site. Journal of the American Chemical Society, 124 (40), 11866-11867. 

Schoch, G.A., Yano, J.K., Wester, M.R., Griffin, K.J., Stout, C.D. and Johnson, E.F. (2004) Structure of human microsomal cytochrome P450 2 C8. Evidence for a peripheral fatty acid binding site. The Journal of Biological Chemistry, 279, 9497-9503. 

Wilson MT, Antonini G, Malatesta F, et al. 1994. Probing the oxygen binding site of cytochrome c oxidase by cyanide. J Biol Chem 269(39) 24114-24119. 

Another type of inhibitor combines with the enzyme at a site which is often different from the substrate-binding site and as a result will inhibit the formation of the product by the breakdown of the normal enzyme-substrate complex. Such non-competitive inhibition is not reversed by the addition of excess substrate and generally the inhibitor shows no structural similarity to the substrate. Kinetic studies reveal a reduced value for the maximum activity of the enzyme but an unaltered value for the Michaelis constant (Figure 8.7). There are many examples of non-competitive inhibitors, many of which are regarded as poisons because of the crucial role of the inhibited enzyme. Cyanide ions, for instance, inhibit any enzyme in which either an iron or copper ion is part of the active site or prosthetic group, e.g. cytochrome c oxidase (EC 1.9.3.1). 

The interaction of an extrinsic membrane protein with a lipid bilayer can also be investigated by energy transfer. The interaction of cytochrome c has attracted much attention, and in an early study by Shaklai et al.(()5> the number of binding sites per red cell was determined. It was shown that an equation analogous to the Stem-Volmer relationship could be derived ... 

---