Centers of Cytochrome c Oxidase

Cu(I) state. It has been proposed that a site of similar structure is present in the enzyme nitrous oxide reductase (96, 97). 

These facts about the cytochrome 03 site, inhibited by CN, can be interpreted in the following way. Cytochrome 03 is EPR silent but has a ground state with an electronic doublet as the lowest energy compo- 

A comparative study of the metal centers in cytochrome c oxidase from several bacterial sources, including Thermus thermophilus and P. denitrificans, using EPR and MCD spectroscopy has established that in both cases cytochrome a is liganded by two histidine oxidases and the Cua center is identical to that in bovine cytochrome c oxidase (105, 106). The properties of the cytochrome Os/Cub dimer have not been established to be identical, although ferrocytochrome 03 is high-spin ferrous, as expected. Recent studies of the MCD properties of the Cua center in cytochrome c oxidase and a copper center in nitrous oxide reductase (107,108) show that the two centers are virtually identical. The evidence from the EPR hyperfine structure of the copper center in nitrous oxide reductase suggests that the center in this enzyme is a mixed-valence Cu(I)/Cu(II) dimer, which raises the interesting prospect that the Cua center in cytochrome c oxidase is also a dimeric copper species. 

We have benefited from discussions with G. R. Moore, R. Grinter, P. Nicholls, M. J. Stillman, C. A. Reed, P. J. Stephens, and A. Walker. 

Keilin, D., in The History of Cell Respiration and Cytochromes (J. Keilin, ed.) Cambridge Univ. Press, Cambridge, 1966. 

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M. Fabian and co-workers have studied the protein s role in internal electron transfer to the catalytic center of cytochrome c oxidase using stopped-flow kinetics. Mitochondrial cytochrome c oxidase, CcO, an enzyme that catalyzes the oxidation of ferrocytochrome c by dioxygen, is discussed more fully in Section 7.8. In the overall process, O2 is reduced to water, requiring the addition of four electrons and four protons to the enzyme s catalytic center. Electrons enter CcO from the cytosolic side, while protons enter from the matrix side of the inner mitochondrial membrane. This redox reaction. 

The biological significance of these reactions is considered further in Chapters 18 and 24. The 132-kDa dimeric N20 reductase from Pseudomonas stiltzeri contains four copper atoms per subunit.546 One of its copper centers resembles the CuA centers of cytochrome c oxidase. A second copper center consists of four copper ions, held by seven histidine side chains in a roughly tetrahedral array around one sulfide (S2 ) ion. Rasmussen et al. speculate that this copper-sulfide cluster may be an acceptor of the oxygen atoms of N20 in the formation of N2.546a There is also a cytochrome cdj type of nitrite reductase.1433... 

Midpoint Potentials fob the Metal Centers of Cytochrome c Oxidase in Isolated Protein and in Mitochondria... 

C. Diheme Cytochrome c Peroxidase Pseudomonas aeruginosa) Metal Centers of Cytochrome c Oxidase... 

Fig. 9. Structure of the CuA-center of cytochrome c oxidase from bovine heart. The gray circles represent the copper ions, the dotted lines are the coordinate bonds between the copper ions and ligands. From Tsukihara et al. 1995 [44] with permission...

The cyclic oxidation and reduction of the iron and copper in the oxygen reduction center of cytochrome c oxidase, together with the uptake of four protons from the matrix space, are coupled to the transfer of the four electrons to oxygen and the formation of water. Proposed Intermediates in oxygen reduction Include the peroxide anion Oz ) and probably the hydroxyl radical (OH-) as well as unusual complexes of iron and oxygen atoms. These intermediates would be harmful to the cell if they escaped from the reaction center, but they do so only rarely. 

It has been isolated as a two-subunit protein, but genetic evidence suggests the presence of additional subunits. The small subunit is a cytochrome c, while the larger subunit is predicted to bind two protohemes as well as a nonheme iron center. This protein also shows sequence homology with cytochrome c oxidase. It contains no copper, but it has been suggested that a heme l)-nonheme Fe center similar to the heme fl-Cug center of cytochrome c oxidase may be presenf. If may be fhe site af which fhe nifrogen atoms of two molecules of NO are joined to form A differenf kind of... 

Recent work has shown NorBC to contain an Fe heme /nonheme Fe binu-clear center, termed NorB, which is both structurally and genomically quite similar to the Fe-Cu center of cytochrome c oxidases. A number of spectroscopic experiments suggest that the diferrous form is required for NO reduction However, the reactivity of a model binuclear heme/nonheme Fe complex suggests that a simple coupling of Fe°-NO species at the two active site metals can yield N2O and an Fe -O-Fe bridging species, and a similar bridging 0x0 species has been observed in the inactive enzyme . 

Figure 18-11 Possible catalytic cycle of cytochrome c oxidase at the cytochrome a3 - CuB site. The fully oxidized enzyme (O left center) receives four electrons consecutively from the cyt c —> CuA —> cyt a chain. In steps a and b both heme a and CuB, as well as the CuA center and cyt a3/ are reduced to give the fully reduced enzyme (R). In the very fast step c the cyt a3 heme becomes oxygenated and in step d is converted to a peroxide with oxidation of both the Fe and Cu. Intermediate P was formerly thought to be a peroxide but is now thought to contain ferryl iron and an organic radical. This radical is reduced by the third electron in step/ to give the ferryl form F, with Cu2+ participating in the oxidation. The fourth electron reduces CuB again (step g) allowing reduction to the hydroxy form H in step h. Protonation to form H20 (step ) completes the cycle which utilizes 4 e + 4 H+ + 02 to form 2 H20. Not shown is the additional pumping of 4 H+ across the membrane from the matrix to the intermembrane space.

Fig. 5. Schematic representation of the interrelationships between the metal centers at the active site of cytochrome c oxidase showing an electronically coupled iron-copper pair (left) and an electronically and magnetically coupled iron-copper pair (right) which interact weakly with each other (—).

The dioxygen reduction site of the key respiratory enzyme, cytochrome c oxidase [E.C. 1.9.3.1], is a bimetallic catalytic center comprised of a heme iron adjacent to a Type 2 mononuclear copper center (see Cytochrome Oxidase). The recent solution of the X-ray crystal structure of this enzyme revealed an entirely unanticipated covalent modification of the protein structure, a cross-link between a histidine and tyrosine side chain (23) within the active site (Figure 2)." This extraordinary posttranslational modification has been confirmed by peptide mapping and mass spectrometry, and has been detected as a conserved element in cytochrome c oxidases isolated from organisms ranging from bacteria to cows. The role of the cross-linked structure in the function of cytochrome c oxidase is still controversial." " ... 

Fig. 12.1. Relative sizes of mitochondrial and chloroplast chromosomes and location of protein structural genes. The figure was constructed from published data [5,15,17,22,26-28]. The structural genes are marked by wide sections. Black areas code for proteins. White areas are introns. 0x1, OxII and OxIII are subunits I, II and III of cytochrome c oxidase. Cyt b, cytochrome b. Fo and Fo, are subunits 6 and 9 of the proton ATPase complex. In the chloroplast chromosome the arrows indicate the transcription direction and the size of the transcripts. CF,a, CFj/8, CFjc and CFoIII are subunits a, /S, t and III of the chloroplast proton ATPase complex [30]. PSII5], PSII44, and PSII34 are subunits of photosystem II reaction center with the corresponding molecular weights of 51000, 44000 and 34000. PSI70 is subunit I of photosystem I reaction center. Cyt /is cytochrome/ cyt is cytochrome b and b -flV is subunit IV of cytochrome b(,-f complex.

CuA-centers are found in cytochrome c oxidases and in N20-reductase [40,41]. In both enzyme classes, CuA-centers subtract electrons from an external donor and transfer them either directly to the active site or indirectly via a further redox-active center [42-44]. Until recently, knowledge concerning the structure of CuA-centers was incomplete. This situation was alleviated by the publication of the crystal-structures of cytochrome c oxidase from Paracoccus denitrificans and bovine heart in 1995 [43,44]. According to these data, CuA-centers contain [2Cu-2S] structures similar to those in [2Fe-2S]-type iron-sulfur clusters. Both sulfur ligands are donated by cysteine residues in the peptide chain and form a planar structure with the copper ions [43-45]. In both structures, an electron can be delocalized over both metal-ions. In the iron-sulfur center this effect is observed in the reduced form [FeZ5+-Fe2 5+], while in the CuA-center the delo-... 

The oxidized form of cytochrome c oxidase contains two Cu and two Fe " heme centers. It can be fully reduced to give a form of the enzyme containing two Cu and two Fe heme centers." " The heme found in cytochrome c oxidase is different from that found in other heme proteins. It is heme a, closely related to heme b, which is found in hemoglobin, myoglobin, and cytochrome P-450, but has one of the vinyl groups replaced by a famesyl substituent and one of the methyl groups replaced by a formyl substituent (see 5.35). 

Schematic representation of the metal centers in cytochrome c oxidase.

A single turnover in the reaction of cytochrome c oxidase involves (1) reduction of the four metal centers by four equivalents of reduced cytochrome c,... 

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