Intermonomer interactions in dimer of bovine heart cytochrome c oxidase.

The X-ray structure of bovine heart cytochrome c oxidase solved for orthorhombic crystals showed a dimeric structure stabilized by four subunit-subunit contacts, namely, subunit Vb-subunit Vb on the matrix side, subunit I-subunit VIa, subunit VIa-subunit I in the transmembrane region and subunit VIb-subunit VIb on the intermembrane side. The same intermonomer contacts as in the orthorhombic crystals were observed in both hexagonal and tetragonal crystals, the X-ray structures of which were determined by the molecular-replacement method. These results suggest that the dimeric structure also exists under physiological conditions. These contacts, especially the subunit IVa-subunit I contact, in which the N-terminal portion of subunit IVa is placed on the surface of subunit I near the dioxygen-reduction site, indicate that the function of the bovine heart enzyme is likely to be controlled by perturbation of the monomer-monomer association.

X-ray structure of azide-bound fully oxidized cytochrome c oxidase from bovine heart at 2.9 A resolution.

Two azide ions were identified, one between the Fe and Cu atoms in the O(2)-reduction site and the other at the transmembrane surface of the enzyme, in the crystal structure of the azide-bound form of bovine heart cytochrome c oxidase at 2.9 A resolution. Two geometries, a mu-1,3 type geometry between the Fe and Cu atoms and a terminal geometry on the Fe atom, are equally possible for an azide ion in the O(2)--reduction site. The other azide molecule was hydrogen bonded to an amide group of an asparagine and a hydroxyl group of tyrosine in a mu-1,1 type geometry. The antisymmetric infrared bands arising from these azide ions, which show essentially identical intensity [Yoshikawa & Caughey (1992), J. Biol. Chem. 267, 9757-9766], strongly suggest terminal binding of the azide to Fe. The electron density of all three imidazole ligands to Cu(B) was clearly seen in the electron-density map of the azide-bound form of bovine heart enzyme, in contrast to the crystal structure of the azide-bound form of the bacterial enzyme [Iwata et al. (1995), Nature (London), 376, 660-669], which lacks one of the three imidazole ligands to Cu(B).

X-ray structure and the reaction mechanism of bovine heart cytochrome c oxidase.

X-ray structure of bovine heart cytochrome c oxidase in the fully oxidized state shows a peroxide bridging between Fe2+ and Cu2+ in the O2 reduction site. The bond distances for Fe-O and Cu-O are 2.52 and 2.16 A, respectively. The structure is consistent with antiferromagnetic coupling between the two metals, which has long been known and to recent redox titration results [J. Biol. Chem. 274 (1999) 33403]. The trigonal planer coordination of Cu1+ in the O2 reduction site is consistent with the very weak interaction between Cu1+ and O2 bound at Fe2+ revealed by time-resolved resonance Raman investigations. One of the three histidine imidazoles coordinated to the Cu ion in the O2 reduction site fixes a tyrosine phenol group near the O2 reduction site with the direct covalent link between the two groups. The structure suggests that the phenol group is the site for donating protons to the bound O2. Redox-coupled conformational change in an extramembrane loop indicates that an aspartate (Asp51) in the loop apart from the O2 reduction site is the site for proton pumping.

Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase.

Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25-1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O(2)-oxidized enzyme. The O(2)-oxidized form, when exposed to excess amounts of O(2), was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O(2)-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O(2)-oxidized form, are due to a bound peroxide produced by O(2) and electrons from the internal donors, consistently with a peroxide at the O(2) reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K. , Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-1729).

Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one.

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.

Structure analysis of bovine heart cytochrome c oxidase at 2.8 A resolution.

The crystal structure of bovine heart cytochrome c oxidase has been determined at 2.8 A resolution by the multiple isomorphous replacement (MIR) method with three heavy-atom derivatives. An asymmetric unit of the crystal has a molecular weight of 422 kDa. Eight heavy atoms as main sites of a CH3HgCl derivative were clearly located by solving the difference Patterson function. The electron density obtained by the MIR method was refined by density modification, consisting of solvent flattening, histogram matching and non-crystallographic symmetry averaging. The enzyme exhibits a dimeric structure in the crystal. Out of 3606 amino-acid residues in 26 subunits in the dimer, 3560 residues were located in the electron-density map. The structure was refined by X-PLOR. The final R factor and the free R factor were 0.199 and 0.252 at 2.8 A resolution, respectively. One monomer in the dimeric structure with a stronger packing interaction has a lower averaged temperature factor than the other, by 16 A2. The region +/-12 A from the centre of the transmembrane part is almost 100% alpha-helix, despite the glycine residue content being as high as 7.1% in the transmembrane region. The residues around haem a of animals have evolved away from those of bacteria in contrast with the residues of the haem a3. The hierarchy of the structural organization of the enzyme complex has been proposed on the basis of intersubunit interactions.

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy.

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.

Crystals of bovine heart ubiquinol-cytochrome c reductase diffracting X-rays up to 2.8 A resolution at 276 K.

Bovine heart ubiquinol-cytochrome c reductase stabilized with sucrose monocaplate was crystallized with polyethylene glycol as the precipitant at 277 K. X-rays diffracted by the crystal were detected up to 2.8 A resolution at 266 K, without using a synchrotron source. The space group and cell dimensions are P61 or P65 and a = b = 128.5 and c = 715.7 A, respectively.

Crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution.

Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 A resolution. A propionate of hems a, a peptide unit (-CO-NH-), and an imidazole bound to CuA are hydrogen-bonded sequentially, giving a facile electron transfer path from CUA to heme a. The O2 binding and reduction site, heme a3, is 4.7 A apart from CuB. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O2 reduction site. The O2 reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of CuB and the location of Tyr244 in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O2 reduction process.

Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase.

Crystal structures of bovine heart cytochrome c oxidase in the fully oxidized, fully reduced, azide-bound, and carbon monoxide-bound states were determined at 2.30, 2.35, 2.9, and 2.8 angstrom resolution, respectively. An aspartate residue apart from the O2 reduction site exchanges its effective accessibility to the matrix aqueous phase for one to the cytosolic phase concomitantly with a significant decrease in the pK of its carboxyl group, on reduction of the metal sites. The movement indicates the aspartate as the proton pumping site. A tyrosine acidified by a covalently linked imidazole nitrogen is a possible proton donor for the O2 reduction by the enzyme.

ATP and ADP bind to cytochrome c oxidase and regulate its activity.

By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.

[Crystal structure of fully oxidized cytochrome c-oxidase from the bovine heart at 2.8 A resolution]. / Kristallicheskaia struktura polnost'iu okislennoi formy thitokhrom c-oksidazy iz serdtsa byka s razresheniem 2,8 A.

Structural and functional studies on cytochrome c oxidase before the crystal structures appeared, are summarized to show the importance of X-ray crystal structure at the atomic resolution for understanding mechanism of the enzyme reaction, O2 reduction coupled with proton pumping. Crystal structure of bovine heart enzyme at the fully oxidized state at 2.8 A resolution are reviewed to evaluate of the contribution for understanding the enzyme reaction mechanism. Effects of detergent structure on the crystallization conditions of the bovine heart cytochrome c oxidase, which was critical for obtaining the X-ray crystal structure, are presented to propose a possible mechanism of crystallization of multicomponent membrane proteins.

Resonance Raman/absorption characterization of the oxo intermediates of cytochrome c oxidase generated in its reaction with hydrogen peroxide: pH and H2O2 concentration dependence.

Effects of pH and H2O2 concentration on the reaction of cytochrome c oxidase (CcO) with H2O2 were studied with the high-performance Raman/absorption simultaneous determination technique reported previously (Proshlyakov et al., 1996). This reaction generates two intermediates called 607- and 580-nm forms, and we found that they show the same oxygen-isotope-sensitive RR bands as those of the intermediates in O2 reduction by CcO. In transient absorption spectra obtained under single turnover conditions, the 607-nm form appeared as the primary intermediate and subsequently the 580-nm and resting forms, suggesting that H2O2 serves as an oxidant for the resting enzyme but as a reductant for both the 607- and 580-nm forms in the peroxide cycle. The rise rate of absorption at 607 nm was insensitive to the H2O/D2O exchange, but the decay was significantly slower in D2O than in H2O. With the microcirculating system, each intermediate was maintained at a constant level under steady-state conditions by supplying H2O2 continuously. In the pH range between 7.4 and 10.0, the population of the 607-nm form decreased at higher pH and at higher concentrations of H2O2. The Fe=O stretching (VFe=O) frequencies of the oxo heme of the 607-nm form, observed at 804/769 cm-1 for their H2(16)O2/H2(18)O2 derivatives, were unaltered in this pH range and exhibited a D2O/H2O shift even at pH 10.0. This indicates that the iron-bound oxygen is hydrogen-bonded to a distal residue in this pH range. When the 580-nm form is dominant under the nonsaturating level of H2O2, two other oxygen-isotope-sensitive Raman bands have been observed at 785/750 cm-1 and 355/340 cm-1 at neutral pH, but the former disappeared above pH 8.5 and the latter above pH 9.0 without significant changes of absorption spectra, suggesting the presence of two separate species in the name of the 580-nm form. However, under the saturating concentration of H2O2, these Raman bands were unaltered between pH 7.4 and 10.0. In contrast, in the absence of excess peroxide, no oxygen-isotope-sensitive RR bands were observed despite dominance of the 580-nm form. The disappearance of these Raman bands demonstrates the occurrence of oxygen exchange between the oxo heme and bulk water, whose rate surpasses the formation rate of the 580-nm form at alkaline pH and/or at low H2O2 concentration. Such an oxygen exchange did not take place in the 607-nm form. Under the identical experimental conditions for generating a particular steady state, the exchange of H2O with D2O caused significant depopulation of the 580-nm form and concomitant increase of the 607-nm form. This was satisfactorily interpreted in terms of the difference in the decay rate of the 607-nm form between H2O and D2O. Thus, the reduction of the 607-nm form to the 580-nm form is likely to be a key step of the redox-linked proton pumping in the O2 reduction.

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

Microcirculating system for simultaneous determination of Raman and absorption spectra of enzymatic reaction intermediates and its application to the reaction of cytochrome c oxidase with hydrogen peroxide.

A new high-performance device for Raman/absorption simultaneous determination was developed. This was combined with a newly designed microcirculating system and was successfully applied to study intermediates in the reaction of bovine oxidized cytochrome c oxidase (CcO) with hydrogen peroxide under steady state conditions at ambient temperatures. Measurements with this device made it possible to correlate directly the species defined in terms of the visible absorption characteristics with specific Raman bands. The "607 nm" form of the enzyme obtained with H2(16)O2 gave an oxygen isotope sensitive band at 804 cm-1 (769 cm-1 with H2(18)O2) in the Soret excited resonance Raman (RR) spectrum. Its frequency and isotope frequency shifts are exactly the same as those observed previously with 607 nm excitation in nonsimultaneous measurements for the 607 nm form, for which the presence of an oxoiron heme was demonstrated. The so-called " 580 nm" form of the enzyme obtained with H2(16)O2 gave the main oxygen isotope sensitive band at 785 cm-1 (750 cm-1 with H2(18)O2) but appeared to consist of multiple species. This band was assigned to the FeIV = O stretching mode of ferryloxo heme on the basis of its isotopic frequency shift. Another oxygen isotope sensitive band was found at 355 cm-1 (340 cm-1 for H2(18)O2), similar to the case of dioxygen reaction. Temporal behavior of this band did not agree with either that of the 804 cm-1 band or that of the 785 cm-1 band but seemed to grow between the two species. The RR spectra in the higher frequency region of the 607 nm and 580 nm forms excited at 427 nm were quite alike and did not support the formation of a porphyrin pi-cation radical.

Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A.

The high resolution three-dimensional x-ray structure of the metal sites of bovine heart cytochrome c oxidase is reported. Cytochrome c oxidase is the largest membrane protein yet crystallized and analyzed at atomic resolution. Electron density distribution of the oxidized bovine cytochrome c oxidase at 2.8 A resolution indicates a dinuclear copper center with an unexpected structure similar to a [2Fe-2S]-type iron-sulfur center. Previously predicted zinc and magnesium sites have been located, the former bound by a nuclear encoded subunit on the matrix side of the membrane, and the latter situated between heme a3 and CuA, at the interface of subunits I and II. The O2 binding site contains heme a3 iron and copper atoms (CuB) with an interatomic distance of 4.5 A; there is no detectable bridging ligand between iron and copper atoms in spite of a strong antiferromagnetic coupling between them. A hydrogen bond is present between a hydroxyl group of the hydroxyfarnesylethyl side chain of heme a3 and an OH of a tyrosine. The tyrosine phenol plane is immediately adjacent and perpendicular to an imidazole group bonded to CuB, suggesting a possible role in intramolecular electron transfer or conformational control, the latter of which could induce the redox-coupled proton pumping. A phenyl group located halfway between a pyrrole plane of the heme a3 and an imidazole plane liganded to the other heme (heme a) could also influence electron transfer or conformational control.

Effects of ethyleneglycol chain length of dodecyl polyethyleneglycol monoether on the crystallization of bovine heart cytochrome c oxidase.

Tetragonal crystals that diffracted X-rays up to 5 A resolution were obtained from bovine heart cytochrome c oxidase isolated and solubilized with dodecyl octaethyleneglycol monoether, CH3(CH2)11O(CH2CH2O)8H. Comparison of observed structure factors between data sets each obtained from a different native crystal gave correlation coefficients of 0.92, 0.84 and 0.57 at 10 A, 7 A and 6 A resolution, respectively. The space group and the cell dimensions of the crystal are I4(1) or I4(3) and a = b = 253 A, c = 507 A, respectively. The perfection and stability of the tetragonal crystals are significantly higher than those of the hexagonal crystals of the protein stabilized with Brij-35, CH3(CH2)11O(CH2CH2O)23H (whose details are reported elsewhere). Examination of the effect of ethyleneglycol chain length on the crystallization revealed that only dodecyl polyethyleneglycol monoethers with eight and seven units were appropriate for producing this type of crystal, indicating an optimum size of the detergent for crystallization of the membrane protein.

New crystal forms and preliminary X-ray diffraction studies of mitochondrial cytochrome bc1 complex from bovine heart.

Two new crystal forms of the cytochrome bc1 complex (ubiquinol:ferricytochrome c oxidoreductase, EC. 1.10.2.2) from bovine heart mitochondria were obtained by the batch method with polyethylene glycol 4000 as the precipitant, when buffer was exchanged from Tris-HCl to potassium phosphate. The first crystal form belongs to the hexagonal space group P61 or P6(5) with unit-cell constants of a = b = 131 A and c = 720 A. The reproducibility of crystallization and the quality of this crystal form were improved by an addition of ZnCl2 to the protein solution. The second crystal form, obtained from the enzyme preparation purified by crystallization, belongs to the tetragonal space group P4(1) or P4(3) with unit-cell constants of a = b = 190 A and c = 445 A. Both crystals diffracted X-rays to 6.5 A resolution and gave sharper diffraction spots than the previous monoclinic form. X-ray intensity data to 8.0 A resolution for the hexagonal crystal and to 7.0 A resolution for the tetragonal one were collected with synchrotron radiation.

Selective resonance Raman observation of the "607 nm" form generated in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide.

Resonance Raman spectra were measured selectively for the "607 nm" form, which had been assigned to a peroxy intermediate formed in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide at ambient temperature. A single oxygen isotope-sensitive band was found at 803 cm-1 for the reaction with H2(16)O2 (at 769 cm-1 with H2(18)O2) upon excitation at 607 nm, the wavelength of the difference absorption maximum characteristic of the "peroxy" intermediate. Upon excitation at shorter wavelengths (down to 580 nm), the Raman spectrum simply became weaker without yielding any new features. When H2(16)O18O was used, two bands were observed at 803 and 769 cm-1 (within an accuracy of 0.5 cm-1), but with only half the intensity of those observed with H2(16)O2 or H2(18)O2, which ruled out the possibility that the 803 cm-1 band arose from the O-O or Fe-O2 stretching of the FeIII(O-O-) heme. Conversely, the 34-cm-1 downshift with 18O is in good agreement with the calculated 16O/18O shift (35 cm-1) expected for the diatomic Fe = 16O oscillator at 803 cm-1. This band exhibited an upshift by 1.3 cm-1 in 2H2O, similar to the case of compound II of horseradish peroxidase at neutral pH, and indicative of the presence of a hydrogen bond to the FeIV = O oxygen. The 803/769 cm-1 pair of resonance Raman bands were also observed upon blue excitation, as is the case for the bands found in the dioxygen cycle of this enzyme (Ogura, T., Takahashi, S., Hirota, S., Shinzawa-Itoh, K., Yoshikawa, S., Appelman, E. H., and Kitagawa, T. (1993) J. Am. Chem. Soc. 115, 8527-8536). This observation provides the first direct characterization of the 607 nm form of this enzyme in its reaction with H2O2.