A

Fig. 4. Stereoviews of the CuA site and the heme a site. The blue cages are composed at 2a of electron density at 2.8 Â resolution together with yellow cages, drawn at 10a of the native anomalous difference Fourier at 4.5 Â resolution. (A) CuA site: The two blue balls show the positions of two copper atoms. (B) Heme a site: The red structure represents the porphyrin plane with a red ball showing the position of iron.

Fig. 4. Stereoviews of the CuA site and the heme a site. The blue cages are composed at 2a of electron density at 2.8 Â resolution together with yellow cages, drawn at 10a of the native anomalous difference Fourier at 4.5 Â resolution. (A) CuA site: The two blue balls show the positions of two copper atoms. (B) Heme a site: The red structure represents the porphyrin plane with a red ball showing the position of iron.

density maps at lower resolution, including those of the fully oxidized enzyme azide derivative at 2.9 A resolution (9,54), the fully reduced enzyme CO derivative at 2.8 A resolution (9), and the fully oxidized enzyme free from the inhibitors at 2.8 A resolution (6), although these electron density maps are not accurate enough to conclude the covalent linkage nonempirically. The covalent linkage is reported for the Paraccocus enzyme at 2.7 A resolution (55).

As shown in Fig. 5, the phenol-ring plane of Tyr244 and the imidazol-ring plane of His240 are not in the same plane to form a significant interaction between the two n-electron systems. The angle between the two planes is about 60°. However, the covalent linkage could be a part of a facile electron-transfer pathway from CuB to the ligand of Fea3 (such as O2 and -OOH), which is hydrogen-

Fig. 5. X-ray structure of the O2 reduction site of bovine heart cytochrome-c oxidase in the fully oxidized state at 2.3 Â resolution. The cages show the \F0-Fc\ difference Fourier map of the oxidized form calculated by omitting His240, Tyr244, and any ligand between Fea3 and CuB from the Fc calculation. The cages are drawn at the 7o level (1o = 0.00456e-/Â3).

Fig. 5. X-ray structure of the O2 reduction site of bovine heart cytochrome-c oxidase in the fully oxidized state at 2.3 Â resolution. The cages show the \F0-Fc\ difference Fourier map of the oxidized form calculated by omitting His240, Tyr244, and any ligand between Fea3 and CuB from the Fc calculation. The cages are drawn at the 7o level (1o = 0.00456e-/Â3).

bonded to the OH group of Tyr244. In the fully reduced form, no ligand is detectable between CuB and Fea3 (9). On reduction of the fully oxidized enzyme, the Fea3-CuB distance increases from 4.91 A to 5.19 A as a result of the movement of CuB relative to the fixed position of Fea3 (9). No other conformational change is induced on reduction in the CuB-Fea3 site. Thus, in the fully reduced state, CuB has a trigonal planer geometry.

Three redox inactive metal sites are detectable in the electron density map of the enzyme at 2.3 A resolution (9). The magnesium site is located on the interface between subunits I and II. The carboxyl groups of Glu198 of subunit II and the two amino acids of subunit I, the imidazole of His368, and the peptide carbonyl group of Asp369 coordinate to the magnesium ion. In addition to these three ligands, three water molecules are coordinated to form a slightly distorted tetragonal bipyramid. The magnesium is located near the level of the mitochondrial innermembrane surface (9). The zinc atom is coordinated by four SH groups of Cys60, Cys62, Cys82, and Cys85 in subunit Vb, which is encoded by a nuclear gene and is attached to subunits I and II on the matrix side (6). A sodium ion site is detectable near the intermembrane surface of subunit I (9). Three peptide carbonyl groups of Glu40, Gly45, and Ser441, a carboxyl group of Glu40, and a water molecule coordinate to the Na+ ion to form a trigonal bipyramid. In the bacterial enzyme, the sodium ion is replaced with calcium and includes an extra negatively charged ligand to balance the charge (55).

The physiological roles of these redox inactive metal sites are unknown. The magnesium and zinc ions have been proposed to be the intrinsic constituents because they were reproducibly found in many samples of purified bovine heart cytochrome-c oxidase (56,57). However, the possibility exists that these metals are bound tightly to contaminant proteins and copurified with cytochrome-c oxidase. The X-ray structure showing specific binding of these metals to the intrinsic protein subunits indicates that these metals are intrinsic constituents of this enzyme. Prior to the solution of X-ray structures, the presence of sodium site had not been suggested. These X-ray structures are good examples of the importance of the determination of the composition of a large multicomponent protein.

5. MECHANISM OF O2 REDUCTION BY CYTOCHROME-C OXIDASE

As described earlier, CuB in the reduced state is in a trigonal planar coordination environment with three histidine imidazole groups (9). Usually, cuprous trigonal planar compounds are very stable and are poor ligand acceptors as well as poor electron donors. Thus, O2 bound at Fea32+ is unlikely to interact with CuB1+. The coordination structure of CuB1+ is highly likely to contribute to the astonishingly high stability of the oxygenated form. On the other hand, the hydroxyl group of Tyr244 is fixed near the O2 reduction site. The shortest distance between the OH group and an atomic model of O2 placed on Fea3 is estimated to be 3.4 A. Considering the possible rearrangement of three-dimensional structure when O2 is introduced to Fea32+ site, the OH group of Tyr244 is placed close enough for formation of a hydrogen bond with the O2 bound at Fea32+. The formation of the putative hydrogen bond between the Tyr244 OH group and the bound O2 is rate limited by the rearrangement of the O2 reduction site. Once the hydrogen bond is formed, the OH group could readily donate a hydrogen atom (a proton + an electron) to the bound O2 to form a hydroperoxo intermediate (Fea33+-OOH) by a two-electron reduction process. Because the two-electron reduction process is very rapid, as described in Section 3.4. (47), the formation of the hydroperoxo intermediate is likely to be rate limited by the rearrangement of the three-dimensional structure of the O2 reduction site for the hydrogen-bond formation between O2 and Tyr244. Thus, the process could be as slow as i1/2 = 0.4 ms (53).

The resultant hydroxyl radical of Tyr244, after formation of the hydroperoxo intermediate, could be readily reduced with an electron equivalent from CuB1+ to produce a deprotonated phenol group of Tyr244, which is ready to accept a proton. Now, CuB is in the cupric state. Thus, it is ready to accept a negatively charged ligands. Then, in the next step, the hydroperoxo group bound to Fea33+ could be deprotonated by transfer of the proton to Tyr244. The resultant peroxide bound to Fea33+ is coordi nated to CuB2+. The oxidation of CuB1+ facilitates the reaction step. Ligand-binding studies of cyanide and azide suggest the presence of a proton-accepting site near the O2 reduction site. The availability of acidic protons could stimulate cleavage of the O-O bond and result in a two-electron abstraction process, possibly from Fea33+ and Fea2+. Thus, once the p-peroxide intermediate is formed, the acidic protons from the proton-accepting site could trigger the O-O bond cleavage. In fact, formation of the second intermediate (P form) next to the oxygenated form is coupled to the oxidation of heme a. Also, the resonance Raman results, as described in Section 3.5., shows that the P form is a ferryl oxide form (53).

The reaction step from the oxygenated form to the P form is essentially a four-electron reduction process. Molecular oxygen (O2) in the oxygenated form must receives four electrons to break the O=O bond to provide two oxides (O2-) and Fea3 in the high oxidation state. This four-electron reduction of O2 essentially at once may be the strategy that the enzyme uses for reduction of O2 to water without the release of active oxygen species. This process produces an Fea3 site in the high oxidation state, where the iron is Fe4+. These high oxidation states of iron are very reactive but are trapped in the porphyrin system. Thus, they are much safer than the active oxygen species produced in the O2-reduction site.

The X-ray structure of the fully oxidized form of bovine heart cytochrome-c oxidase as prepared has a peroxide bridging CuB2+ and Fea33+ (9). The stability of this form indicates that no acidic protons is available in the CuB2+-Fea33+ site in the state. The reactivity with cyanide (14) shows that the fully oxidized form as prepared is the "fast" form, which is considered to be an active enzyme form involved in the enzymic turnover (58). However, the X-ray structure suggests that this form is a resting oxidized form and that the bridging peroxide prevents the CuB2+-Fea33+ site from being exposed spontaneously to O2, which is likely to form active oxygen species by extracting electrons from the dimetalic site. It should be noted that no experimental evidence has been obtained for involvement of the "fast" form in the catalytic turnover. On the other hand, two water (OH/H2O) molecules are assigned to the electron density between CuB2+ and Fea33+ of the fully oxidized form of the P"r"coccus enzyme at 2.7 A resolution (55). The electron density map of the fully oxidized form of bovine heart enzyme at 2.8 A resolution is not accurate enough for differentiating peroxide from two water (OH-/H2O) molecules.

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