Electron Transfer from Copper to Heme

For electron transfer from copper to heme, two dominant sets of pathways of comparable efficiency were predicted. In each set of pathways, the point of intermolecular electron transfer was from the backbone O of Glu i of amicyanin to the backbone N of Gly of cytochrome c-551i, and the entry of electrons to iron occurred either via the porphyrin ring or the His ligand. In one set of pathways the exit of electrons from copper occurred via the Cys copper ligand, and the phenolic side chain of Tyr was an intermediate between Cys and Glu. In the other set of pathways the exit of electrons from copper occurred via the Met copper ligand, and the backbone of Lys was an intermediate between Met and Glu i... 

Davidson, V. L., and Jones, L. H., 1996, Electron transfer from copper to heme within the methylamine dehydrogenase-amicyanin-cytochrome C-55H complex. Biochemistry 35 8120n8125. 

The electron transfer reaction from copper to heme within the ternary protein complex was also studied in solution by stopped-flow spectroscopy. Analysis by Marcus theory of the temperature dependence of the limiting first-order rate constant for the redox reaction (Davidson and Jones, 1996) yielded values for the of 1.1 eV and H b of 0.3 cm , and predicted an electron transfer distance between redox centers which was consistent with the distance seen in the crystal structure. Thus, the electron transfer event is rate-limiting for this redox reaction. Experiments are in progress to determine the validity of the predicted pathways for electron transfer shown in Figure 7. 

Yoshikawa et al made the important statement that the role of heme a may be the biggest mystery in the stmcture and function of cytochrome c oxidase . It is indeed not at all obvious why all heme-copper oxidases have a low-spin heme group very close to the binuclear heme-copper site, especially since direct electron transfer from Cua to that site (if it occurred) would take place across almost the same distance. In the cytochrome c oxidases such direct electron transfer is effectively prevented, perhaps due to the bound Mg, the coordination sphere of which lies on the shortest path between... 

As described previously, the ratio of copper to cytochrome a is always 1 and the oxidase activity is inhibited about 50% by the addition of a copper chelating agent. From the above results, it is certain that the copper in cytochrome a as well as the heme iron is an important component in the oxidase reaction. Recently it was shown that the copper in oxidized cytochrome a is in the cupric state and that it changes in valency together with the iron during electron transfer from substrate to oxygen. 

Fig. 13. Schematic representation of a possible mechanism for electron transfer from cytochrome c to the active site of cytochrome c oxidase. The pathway is represented as involving copper as a mediator between the heme edge of cytochrome c and the stacked 2-alkyl chain of heme A followed by transmittal of the electron through the ir system of heme A to dioxygen bound at iron.

The cytochrome c oxidase protein is thought to consist of two heme iron centers (heme a with two axial histidines and heme 03 with one axial histidine (analogous to myoglobin)) and two copper centers (Cua with two histidine, two cysteine, and one water/tyrosine ligand in its oxidized state and Cub with three histidine, one methionine, and one H2O/HO" ligands). The CuA/heme a pair constitute two coupled, one-electron redox couples (low potential, 0.4V) that facilitate (a) electron transfer from cytochrome c(Fe ) at the matrix side of the inner mitochondrial membrane as well as (b) proton transfer from the mitochondrial matrix across the inner membrane to the cytosol. At the cytosol side of the inner mitochondrial membrane, the CuB/heme a- pair constitute the binding site for O2 as well as the conduit for its high-potential four-electron, four-proton reduction to two H2O molecules. 

Cytochrome c oxidase (COX) is the terminal enzyme in the respiratory system of most aerobic organisms and catalyzes the four electron transfer from c-type cytochromes to dioxygen (115, 116). The A-type COX enzyme has three different redox-active metal centers A mixed-valence copper pair forming the so-called Cua center, a low-spin heme-a site, and a binuclear center formed by heme-fl3 and Cub. The Cua functions as the primary electron acceptor, from which electrons are transferred via heme-a to the heme-fl3/CuB center, where O2 is reduced to water. In the B-type COX heme-u is replaced by a heme-fo center. The intramolecular electron-transfer reactions are coupled to proton translocation across the membrane in which the enzyme resides (117-123) by a mechanism that is under active investigation (119, 124—126). The resulting electrochemical proton gradient is used by ATP synthase to generate ATP. 

The kinetics of electron transfer reactions between spinach plastocyanin and [Fe(CN)6] ", [Co(phen)3] , and Fe(II) cytochrome c have been studied as a function of ionic strength. Applications of the equations of Van Leeuwen support the proposal of two sites of electron transfer, with [Co(phen)3] binding near residues 42-45 and the interaction of [Fe(CN)6] at a hydrophobic region near the copper ion. Pulse radiolysis has been employed to measure the rates of electron transfer from Ru(II) to Cu(II) in plastocyanins from Anabaena variabilis and Scenedesmus obliquus which have been modified at His-59 by [Ru(NH3)5] . The small intramolecular rates (<0.082 and <0.26 s , respectively) over a donor-acceptor distance of 12 A indicate that electron transfer from the His-59 site to the Cu center is not a preferred pathway. A more favorable route, via the acidic (residues 42-44) patch ( 14 A to Cu), is supported by the rate of >5 x 10 s for the reduction of PCu(II) by unattached [Ru(NH3)5im] . The intramolecular electron transfer from Fe(II) in horse cytochrome c to Cu(II) in French bean plastocyanin ( 12 A from heme edge to Cys-84 S), in a carbodiimide cross-linked covalent complex, proceeds with a rate of 1.05 x 10 s . The presence of the... 

Both crystallographic studies of nitrite binding to the oxidized enzyme (12) and studies of Type 2 Cu-depleted enzyme (24), where a linear correlation between Type 2 Cu content and specific activity was observed, have shown that the Type 2 Cu center is the site at which NO2 binds and is reduced, analogous to the heme center in the heme cdi enzymes. The role of the Type 1 Cu appears to be that of an electron transfer center, analogous to the role of the heme c in the heme cd enzymes. Although a wide variety of copper contents, colors, and quaternary structures has been reported for Cu NiR s, it seems likely that most, if not all, of the enzymes have structures and optimal copper contents similar to that of the A. cycloclastes enzymes. This assertion is based on (i) extensive sequence similarities between the A. cycloclastes NiR and that from R aureofaciens, previously reported to contain only a single Type 1 Cu per monomer (11) (ii) on studies of the Cu content of the A. xylosoxidans NiR, also previously reported to contain only Type 1 Cu (1) and (iii) on the extensive immunological cross-reactivity observed with antibodies to the A. cycloclastes enzyme (7). 

For the cytochrome c-plastocyanin complex, the kinetic effects of cross-linking are much more drastic while the rate of the intracomplex transfer is equal to 1000 s in the noncovalent complex where the iron-to-copper distance is expected to be about 18 A, it is estimated to be lower than 0.2 s in the corresponding covalent complex [155]. This result is all the more remarkable in that the spectroscopic and thermodynamic properties of the two redox centers appear weakly affected by the cross-linking process, and suggests that an essential segment of the electron transfer path has been lost in the covalent complex. Another system in which such conformational effects could be studied is the physiological complex between tetraheme cytochrome and ferredoxin I from Desulfovibrio desulfuricans Norway the spectral and redox properties of the hemes and of the iron-sulfur cluster are found essentially identical in the covalent and noncovalent complexes and an intracomplex transfer, whose rate has not yet been measured, takes place in the covalent species [156]. 

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