Cytochrome heme crevice

The reaction between HiPIP and cytochrome c or bacterial cytochromes784 involves specific sites on both proteins, although no kinetic evidence for association was found. Reaction between Chr. vinosum and Rhodopseudomonas gelatinosa HiPIPs with modified cytochrome c (trinitro-phenyllysine-13)785 was more rapid than for the unmodified cytochrome c since modification of Lys-13 destabilizes the heme crevice, and because the hydrophobic TNP group facilitates electron transfer by interacting with a hydrophobic region of the HiPIP. 

The pH dependence of cytochrome c oxidation-reduction reactions and the studies of modified cytochrome c thus demonstrate that the coordination environment of the iron and the conformation of the protein are relatively labile and strongly influence the reactivity of the metallo-protein toward oxidation and reduction. The effects seen may originate chiefly from alterations in the thermodynamic barriers to electron transfer, but the conformation changes are expected to affect the intrinsic barriers also. One such conformation change is the opening of the heme crevice referred to above. The anation and Cr(II) reduction studies provide an estimate of 60 sec 1 for this process in Hh(III) at 25°C (59). To date, no evidence has been found for a rapid heme-crevice opening step in ferrocytochrome c. 

Figure 5 A schematic diagram of horse heart c)ftochrome c viewed from the front of the heme crevice. The approximate positions of the /S-carbon atoms of the lysine residues are indicated by closed and dashed circles for residues located toward the front and back of Cc, respectively. The electrostatic free energy contribution of lysine i, Vi, is indicated by the number of diagonal marks in the circle, with —0.4kJM per hatch mark. The binding domain is essentially the same for CcO and cytochrome bc ...

Mitochondrial cytochrome c is perhaps the most widely studied of all metalloproteins with respect to its electrochemical properties. It is located in the inner-membrane space of mitochondria and transfers electrons between membrane-bound complex III and complex IV. The active site is an iron porphyrin with a redox potential (7) of -1-260 mV vs. NHE. The crystal structures of cytochrome c from tuna have been determined (8, 9) in both oxidation states at atomic resolution. It is found that the heme group is covalently linked to the protein via two thioether bridges, and part of its edge is exposed at the protein surface. Cytochrome c is a very basic protein, with an overall charge of -1-7/-l-8 at neutral pH. Furthermore, many of the excess basic lysine residues are clustered around the mouth of the heme crevice, giving rise to a pronounced charge asymmetry. 

The eight critical aromatic groups are not distributed at random over the molecule but tend to occur in pairs. Tyrosine-48 and phenylalanine-tyrosine-46 are found at the bottom of the heme crevice, where 48 helps to tie down the heme and 46 provides a continuation of the hydro-phobic patch formed by the edge of the heme. These residues are also present in identical positions in bacterial cytochromes Cj and C550. Phenylalanine-10 and tyrosine-phenylalanine-97 occur at the right of the molecule, in the midst of a region of packed hydrophobic side chains which has been called the right channel is important to remem-... 

The last of the critical eight aromatic rings is phenylalanine-82, totally invariant over all 60 eukaryotic sequences and the two bacterial cytochromes whose three-dimensional structures are known. It is found nested against the heme, closing the upper left of the heme crevice as shown in Fig. 6. Although this residue was formerly believed from the two-derivative, 2.8 A resolution electron density map of horse oxidized cytochrome to be swung out and away from the heme in ferricytochrome (12-1J ), the four-derivative, 2.0 A map of tuna ferricytochrome has revealed this to be incorrect (15). In both oxidation states, phenylalanine-82 appears to lie next to the heme, and the crevice remains "closed in the sense observed in Fig. 7. 

In the first area we have gone a step backward recently in order to make two steps forward. The reduced tuna cytochrome map at 2 A resolution is in good shape, the oxidized horse cytochrome map at 2.8 A is of poor quality, and the oxidized tuna map at 2 A that will replace it is completed but not interpreted at the time of writing. The opening of the heme crevice at phenylalanine-82 in the oxidized state that was reported from the horse map (12) apparently is not correct, and any comments about the finer reorientations of aromatic or other side chains must wait for confirmation from the new tuna map. Much can be said now about areas 2 through 5 above, however, to lay the groundwork for a later discussion of the first area. 

It should be kept in mind that the cytochrome c molecule is about 30 A in diameter, and this is approximately the diameter of one of the cylindrical Fab arms of the antibody molecule. A bound Fab fragment probably would block an entire quadrant of the surface of the cytochrome molecule. It could bind to lysine-72 and still block the edge of the heme crevice from approach by oxidase or reductase. 

Fig. 21. Forehead view of reduced tuna cytochrome c, looking directly into the heme crevice. Lysine or arginine residues on the front face of the tuna cytochrome molecule are indicated by double circles. The six heavy black circles mark front-side residues which are lysine or arginine in 56 or more of the 60 known sequences of Table IX.

Fig. 22. Corresponding view of R. rubrum c.. Heavy black circles locate lysines and arginines surrounding the heme crevice in R. rubrum Ci. Double circles mark front-side lysines and arginines in one or more of the other three Cs s of Table VII. Only groups in the immediate vicinity of the heme crevice are so marked, and lysines on the extra loops not shared with cytochrome c are also unmarked.

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. 

Fio. 24. Schematic view of the heme crevice with highly conservative residues marked (+) basic, (—) acidic, (Ar) aromatic, (Q) glutamine, and (F) phenylalanine. Residue numbering is that of tuna cytochrome. 

Figure 3 X-ray crystal structure of horse heart cytochrome c. Lysine residues surrounding the heme crevice are blue, and the heme is red...

X 10 M s and was 3.1 x 10 M s" at 25°C, pH 7.0 and ionic strength of 1.0 . Kinetic data was interpreted in terms of a mechanism of electron transfer from chromium(II) involving attack of Cr(II) adjacent to the Fe(III) center Analysis of the one-to-one chromium(III) cytochrome c complex revealed that the chromium(III) cross-linked two peptide fragments located in the heme.crevice by binding to tyrosine 67 and asparagine 52 The chromium(III) bound to reduced cytochrome c did not affect the ability of the protein to be reoxidized with ferricyanide and then to be reduced with dithionite . The chromium complex was oxidized by cytochrome oxidase at the same rate as the untreated ferrocytochrome c, however, the rate of reduction of the chromium complex by bovine heart submitochondrial particles was slower than that of untreated ferricytochrome c Thus, the binding of chromium(III) to cytochrome c appears to selectively inhibit its function in certain electron transfer reactions. 

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