Infrared and EPR studies on cyanide binding to the heme-copper binuclear center of cytochrome bo-type ubiquinol oxidase from Escherichia coli. Release of a CuB-cyano complex in the partially reduced state.

Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a 12C-14N stretching vibration at 2146 cm-1 characteristic of the FeO3+ C=N CuB2+ bridging structure was quickly replaced with another stretching mode at 2034.5 cm-1 derived from the FeO2+ C=N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm-1. This 2169 cm-1 species could not be retained with a membrane filter (molecular weight cutoff = 10,000) and showed unusual cyanide isotope shifts and a D2O shift. These observations together with metal content analyses indicate that the 2169 cm-1 band is due to a CuB.CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the CuB center can be easily released upon cyanide-binding.

across the cytoplasmic membrane not only by scalar protolytic reactions at the inner and outer surfaces of the membrane but also by a proton pumping mechanism (3)(4)(5). Based on the structural homologies of subunits I, II, and III, cytochrome c oxidases and some bacterial quinol oxidases including the E. coli cytochrome bo are classified in the heme-copper respiratory oxidase superfamily (6,7). However, there is a notable difference in electron-donating substrates between the two enzymes, cytochrome c (a hydrophilic one-electron carrier) and quinols (hydrophobic two-electron and two-proton carriers) (8,9). As a consequence, the Cu A center is absent in subunit II of quinol oxidase (5,10,11).
Although the dioxygen reduction mechanism at the hemecopper binuclear center is thought to be identical in both enzymes (12)(13)(14), these differences raise the questions whether the electron transfer reactions from the substrates to dioxygen and the proton pumping mechanism coupled to these redox reactions are alike or distinct. It is proposed that the last two steps of the four-electron transfer reactions to dioxygen are linked to proton pumping by cytochrome c oxidase (15)(16)(17). Thus it becomes increasingly important to analyze the mixedvalence states of oxidase as a model for the intermediate species of the dioxygen reduction chemistry, since the understanding of the redox-linked structural change(s) at the metal center appears to be a key point to reveal the redox-linked proton pumping.
In a previous study, Tsubaki (18) analyzed cyanide binding to the Fe a3 -Cu B binuclear center of cytochrome c oxidase. In the resting (air-oxidized) state, a bound cyanide showed an infrared C-N stretching band at 2152 cm Ϫ1 , assignable to a bridging structure, Fe a3 3ϩ -CϭN-Cu B 2ϩ . This assignment was confirmed recently by structural characterizations and infrared measurements on a series of model complexes containing the [Fe 3ϩ -CϭN-Cu 2ϩ ] bridge unit (19). Upon partial reduction of the CN-inhibited cytochrome c oxidase an infrared band appeared at 2131 cm Ϫ1 assignable to the Fe a3 3ϩ -CϭN structure. Further reduction resulted in an appearance of two new infrared bands at 2058 and 2045 cm Ϫ1 , concomitantly, assignable to the Fe a3 2ϩ -CϭN species. These observations suggest three kinds of conformational change to occur at the Fe a3 -Cu B binuclear site as the reduction of the metal centers proceeds (18).
Subsequently Tsubaki et al. (11) carried out a combined study using EPR and FT-IR spectroscopies to clarify the structural differences of the binuclear center between bo-type ubiquinol oxidase and cytochrome c oxidase. EPR spectra of bo-type ubiquinol oxidase in the air-oxidized state showed EPR signals from an integer spin system confirming the existence of the spin-spin exchange-coupled binuclear site (20 -23). EPR spectra of the cyanide, azide, and formate complexes in the air-oxidized state indicated that a gross conformation at the binuclear site seems well conserved among the heme-copper oxidase superfamily (11). FT-IR spectroscopy confirmed these observations: the cyanide that binds to the air-oxidized enzyme exhibits an infrared band at 2146 cm Ϫ1 characteristic to the Fe O 3ϩ -CϭN-Cu B 2ϩ structure (11). In the present study we extended the FT-IR and EPR spectroscopic studies to clarify the structure at the heme-copper binuclear center using cyanide as a monitoring probe.
Purification of Cytochrome c Oxidase-Cytochrome c oxidase was isolated from bovine heart using the method of Yoshikawa et al. (26). The crystalline sample was solubilized in 50 mM sodium phosphate buffer (pH 7.4) and was treated with 10 mM EDTA as described previously (18). The sample was, then, diluted with 50 mM Tris-DCl (pD ϭ 8.0) (D 2 O buffer) and was concentrated with a Diaflow apparatus. This treatment was repeated several times for the complete exchange of the medium from H 2 O to D 2 O. For some measurements, cytochrome c oxidase in an H 2 O buffer (50 mM sodium phosphate buffer; pH 7.4) was used.
Measurement of Fourier Transform Infrared and Optical Spectra-FT-IR spectra of the purified bo-type ubiquinol oxidase were measured at 10°C as described previously (11,27). A nominal spectral resolution of 4.0 cm Ϫ1 was chosen. Absolute optical spectra of the oxidase in the infrared cells were measured at room temperature with a UVIKON 860 UV-visible spectrophotometer (Contron Instr.) before and after FT-IR measurements. Partially reduced CN-inhibited enzymes were prepared according to Yoshikawa ad Caughey (28) with slight modifications as described previously (29).
Measurement of EPR Spectra-EPR spectra were measured at 5 or 15 K at X-band (9.23 GHz) microwave frequency with a home-built EPR spectrometer as described previously (11) and a Varian E-12 EPR spectrometer equipped with an Oxford flow cryostat (ESR-900).
Miscellaneous-Metal contents were determined by inductively coupled plasma atomic emission spectrophotometry with an SPS 1200VR plasma spectrometer (Seiko Instruments Inc., Tokyo). Heme contents were analyzed as described previously (30). Bound Q 8 in the purified oxidase was estimated on reverse-phase high performance liquid chromatography analysis as described previously (25). The following potassium cyanide isotopes were used: K 12 C 14 N (natural abundant, Nacalai Tesque); K 12 C 15 N (99.4 atom % 15 N, Isotec Inc.); K 13 C 14 N (99 atom % 13 C, Isotec Inc.); K 13 C 15 N (99 atom % 13 C, 99 atom % 15 N, Icon). Other chemicals were commercial products of analytical grade.  (Fig. 1a). This band showed isotopic shifts (Table I) as expected from a diatomic harmonic oscillator model. Addition of excess sodium dithionite to the air-oxidized CN-inhibited enzyme resulted in a rapid full reduction of metal centers on the basis of visible absorption spectroscopy (data not shown). Simultaneously, the bridging cyanide band at 2146 cm Ϫ1 was replaced with a broad band at 2034.5 cm Ϫ1 (Fig. 2a). Upon cyanide isotopic substitution, this band displayed a similar shift pattern with that of the 2146 cm Ϫ1 band (Table I). This infrared band was assigned to a stretching vibration of the Fe O 2ϩ -CϭN adduct on the basis of the disappearance of this band in the presence of carbon monoxide (CO) (Fig. 2, b and c -CϭN structure showing a 2131 cm Ϫ1 infrared band was observed for cytochrome c oxidase (18).

FT-IR and EPR Spectra of the CN-inhibited bo-type
EPR spectra of the air-oxidized CN-inhibited ubiquinol oxi-  dase and its dithionite-treated (and quenched at 77 K just after the addition) forms were examined at 15 K. Addition of cyanide to the air-oxidized enzyme reduced the intensity of a g ϭ 6 high spin signal without affecting the g ϭ 3 low spin signal (g ϭ 2.98, 2.26, and 1.45) (Fig. 3, a and b) as described previously (11,30). Addition of excess sodium dithionite to the air-oxidized CNinhibited enzyme caused a rapid disappearance of the g ϭ 3 low-spin signal and an appearance of a new low-spin signal with g z ϭ 3.24 (Fig. 3c). Prolonged incubation with sodium dithionite eventually eliminated the g z ϭ 3.24 low spin signal. This EPR signal is assignable to the Fe O 3ϩ -CϭN species (22, 23) on the basis of similarity to the corresponding species (g z ϭ 3.58) of the partially reduced CN-inhibited cytochrome c oxidase (32). Occasionally we observed a weak stretching band at 2123 cm Ϫ1 (for 12 C 14 N) which shifted to 2078 cm Ϫ1 upon 13 C 14 N substitution (Table I). This band also appeared in the partially reduced CN-inhibited states (one-fourth-reduced and one-half-reduced states; see later) and, therefore, may be assignable to the Fe O 3ϩ -CϭN species.

Influences of Loosely Bound Q 8 on the FT-IR Spectra of the Air-oxidized CN-inhibited Ubiquinol
Oxidase-Our ubiquinol oxidase preparations usually contain one to two molecules of loosely bound Q 8 and one tightly bound Q 8 at the Q H site (25). Precipitation of the enzyme with PEG 4000 removes the loosely bound Q 8 from the preparation (25). In contrast to the untreated enzyme that showed a substantial autoreduction, no visible spectral change was observed for the PEG-treated enzyme during the anaerobic incubation with cyanide in the infrared cell at 4°C (spectra not shown). FT-IR spectroscopy confirmed no further spectral change to occur for the PEGtreated oxidase (Fig. 1, a and b) except for decrease in the intensity of the 2093 cm Ϫ1 band of free H 12 C 14 N. Addition of 4-fold excess Q 1 to the PEG-treated enzyme restored the autoreducibility of the metal centers (spectra not shown).
The FT-IR spectra of these autoreduced CN-inhibited enzymes were characterized with the appearance of a strong cyanide stretching band at 2169 cm Ϫ1 (in an H 2 O buffer) (Fig.  1, c and d). Careful measurements on the time-dependent spectral change showed the following. 1) There is a preceding phase forming a 2198 cm Ϫ1 band species (Fig. 1c).
2) The formation of the 2169 cm Ϫ1 band species follows. This phase also shows weak infrared bands at 2076 and 2038 cm Ϫ1 (Fig. 1d). 3) The development of the 2169 cm Ϫ1 band intensity reaches a plateau as the 2146 cm Ϫ1 band disappears, but the 2034.5 cm Ϫ1 band characteristic to the Fe O 2ϩ -CϭN adduct does not grow so strong. 4) A further incubation causes a gradual decrease in the intensity of the 2169 cm Ϫ1 band itself. One of unique features of this 2169 cm Ϫ1 species is its peculiar cyanide-isotopic shift pattern that is very different from the usual metal-bound cyanide species (Table I). The other is its strong D 2 O shift (Table  I). In a D 2 O medium, the 2169 cm Ϫ1 band shifted to 2161 cm Ϫ1 . Further, this 2169 cm Ϫ1 species passed through a membrane filter (MWCO ϭ 10,000) ( Fig. 1e) indicating that this is a small molecular complex released from the enzyme.

Influences of Partial and Full Reduction of the CN-inhibited Ubiquinol
Oxidase-Anaerobic addition of one-or two-electron equivalent(s) (assuming that E. coli bo-type ubiquinol oxidase can accommodate up to five-electron equivalents at a low spin heme B, a high spin heme O, Cu B , and a tightly bound Q 8 (Q H ) (25) 3 to the air-oxidized CN-inhibited enzyme produced rapidly the 2169 cm Ϫ1 band together with bands at 2122, 2093, and 2077 cm Ϫ1 with weaker intensities (spectra not shown). Anaerobic addition of sodium dithionite to the partially reduced CNinhibited oxidase did not affect the 2169 cm Ϫ1 species. Addition of excess sodium dithionite to the oxidase in the presence of Q 1 (or loosely bound endogenous Q 8 ) prior to addition of cyanide did not produce the 2169 cm Ϫ1 band at all. This observation suggests that the partially reduced conditions of the oxidase are essential for the formation of the 2169 cm Ϫ1 species.
Cyanide Binding to the Binuclear Center Mutant and to the Bound Q 8  binuclear center mutant oxidases, H284A, H333A (24,30,33), and Y288L, 2 were examined by FT-IR spectroscopy. When cyanide (5 mM) was added to the air-oxidized H284A mutant oxidase, there was a weak infrared band at 2124 cm Ϫ1 but no band around 2146 cm Ϫ1 . Simultaneous addition of 4-fold excess Q 1 to the mutant oxidase did not cause any infrared spectral change. Addition of cyanide to the Cu B -deficient H333A oxidase caused the appearance of weak infrared bands at 2160 and 2121 cm Ϫ1 , but not at 2169 cm Ϫ1 (spectra not shown). Addition of cyanide to the air-oxidized Y288L mutant oxidase showed no bound cyanide band (spectra not shown). In the dithionitereduced states, these mutant oxidases did not show any clear cyanide band associated with the ferrous heme (spectra not shown). Addition of cyanide to the bound Q 8 -free wild-type oxidase showed a weak infrared band at 2146 cm Ϫ1 together with multiple bands around 2126 cm Ϫ1 region (Fig. 4a). Simultaneous addition of 4-fold excess Q 1 with cyanide gave the same result. Prolonged incubation produced the 2169 cm Ϫ1 band, but it was very weak (Fig. 4b).
Partial Reduction of the CN-inhibited Cytochrome c Oxidase-Upon addition of cyanide (5 mM) to the resting (airoxidized) cytochrome c oxidase in a D 2 O buffer, a cyanide band appeared at 2152 cm Ϫ1 (Fig. 5a), which has been assigned to the Fe a3 3ϩ -CϭN-Cu B 2ϩ species (18). Introduction of a first and a second electron equivalent to the air-oxidized CN-inhibited enzyme caused a dramatic decrease of the 2152 cm Ϫ1 band intensity with a concomitant increase in intensity of a band at 2131 cm Ϫ1 (Fig. 5, b and c), which has been assigned as the Fe a3 3ϩ -CϭN species (18). Introduction of a third electron equivalent removed the 2151 cm Ϫ1 band completely while the 2131 cm Ϫ1 band became further intensified (Fig. 5d).
During these spectral changes we observed an appearance of a 2162 cm Ϫ1 band in a D 2 O buffer (Fig. 5b and c). In a similar experiment carried out in an H 2 O buffer, the 2162 cm Ϫ1 band in the partially reduced states (one-fourth and one-half) shifted to 2169 cm Ϫ1 without affecting the 2152 and 2131 cm Ϫ1 bands (spectra not shown). These observations strongly suggest that a cyanide species very similar to that found in the CN-inhibited bo-type ubiquinol oxidase was produced in the partially reduced state(s) of the CN-inhibited cytochrome c oxidase. The intensity of the 2169 cm Ϫ1 band (in the D 2 O buffer, or the 2162 cm Ϫ1 band in the H 2 O buffer) changed in a dose-and time-dependent manner. A higher cyanide concentration (20 mM) and a longer incubation time (70 h) in the partially reduced state (one-fourth-reduced) caused a stronger intensity of the 2169 cm Ϫ1 band (spectra not shown). The 2165 cm Ϫ1 band (in a D 2 O buffer) observed by Yoshikawa and Caughey (28,34) is likely due to the same species observed in the present study.
Metal Content Analysis-The filtrate that passed through a membrane filter (MWCO ϭ 10,000) after the cyanide treatment of ubiquinol oxidase had no color indicative of a heme species. Therefore, the most reasonable candidate for the 2169 cm Ϫ1 species is a copper-cyano species. A preliminary metal content analysis of the filtrate after the cyanide treatment showed a substantial amount of copper ions; whereas the filtrate without the cyanide treatment shows a trace amount of copper ions (data not shown). To establish a loss of the Cu B center from bo-type ubiquinol oxidase by the cyanide treatment in the presence of ubiquinone, we carried out metal content analyses (Table II). The wild-type oxidase that contained 2.21 mol of Q 8 /mol of the enzyme lost about 30% of the Cu B center upon cyanide treatment, whereas the bound Q 8 -free oxidase retained copper completely.

Implication for the Cyanide Coordination Structure at the
Binuclear Center-The CN-bridging band at 2146 cm Ϫ1 for the air-oxidized CN-inhibited enzyme (11) was 6 cm Ϫ1 lower than that of cytochrome c oxidase (18), probably reflecting a slight increase in a Cu-NC bond and decrease in a Cu-N-C bond angle (19). But the Fe O 2ϩ -CϭN stretching frequency at 2034.5 cm Ϫ1 in the fully reduced state was much lower (23.5 or 11 cm Ϫ1 ) than the corresponding frequencies of cytochrome c oxidase (2058 and 2045 cm Ϫ1 ) (18). The C-O stretching frequency of bo-type ubiquinol oxidase (1959.7 cm Ϫ1 ) (11, 24) was, however, only 3.8 cm Ϫ1 lower than the corresponding frequency of cytochrome c oxidase (1963.5 cm Ϫ1 ) (35). This small change is attributable mostly to the absence of a formyl group in heme O, as suggested before (11).
The large difference of the bound C-N stretching vibration between bo-type ubiquinol oxidase and cytochrome c oxidase in the reduced state is likely due to a specific character of the cyanide-binding to the binuclear center (36). Cyanide binding to other typical ferrous hemoproteins is extremely weak, except for horseradish peroxidase in which the electrostatic interaction (or hydrogen bond) between a protonated distal His residue and a ferrous heme-bound cyanide plays a substantial role in the stabilization (37). Thus, it is possible that the ferrous heme-bound cyanide at the binuclear center is stabilized by a protonated His residue in the vicinity of the heme in the fully reduced state. The protonation of the His residue may be directly coupled to the uptake of a proton upon binding of cyanide to the reduced oxidase (38). Among three invariant His residues (His-284, His-333, and His-334) on the distal side of the high spin heme (6, 7), His-284 is likely to have such a role since it is probably not an obligatory ligand to Cu B unlike His-333 and His-334 (12,24,30). Alternatively, His-333 may perform such a part in cytochrome c oxidase, since it seems to be disordered or to have multiple conformations in an x-ray crystal structure for the azide-inhibited air-oxidized cytochrome c oxidase from Paracoccus denitrificans (39), but not for the air-oxidized cytochrome c oxidase from bovine heart mitochondria (40). The x-ray crystal structures revealed also that His284 can form hydrogen bond with Tyr-288 and Trp-280 and His-333 with Thr-352 or the carbonyl oxygen of Phe-348 (39,40). Thus the greater difference in the Fe 2ϩ -CϭN stretching vibration is likely due to the difference in the interaction between the protonated distal His residue and the Fe 2ϩ -CϭN moiety.
The binuclear center mutant oxidases showed neither CNbridging infrared band in the air-oxidized state nor Fe 2ϩ -CϭN infrared band in the fully reduced state, although several Fe 3ϩ -CϭN species seemed to be formed. It is clear that presence of the Cu B center is essential for the binding of cyanide to the ferrous heme since these mutant oxidases did somehow bind CO (although with a very broad infrared band around 1970 cm Ϫ1 (His333Ala (24) and Y288L) 2 or with very weak affinity (H284A)). However, we could not evaluate the specific role of the imidazole group of His-284 and His-333 and the phenol group of Tyr-288 in the present study.
Structural Implication on the 2169 cm Ϫ1 Species-We found that the presence of excess Q 8 or Q 1 in the wild-type ubiquinol oxidase preparation caused a gradual development of a new cyanide band at 2169 cm Ϫ1 associated with the autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme. The 2169 cm Ϫ1 band could not be observed for the binuclear center mutant oxidases (H284A, Y288L, and H333A) in which the Cu B center was greatly perturbed or almost eliminated (30,33). 2 This 2169 cm Ϫ1 species showed unusual cyanide isotope shifts and a D 2 O shift (Table I) and was able to pass through a membrane filter (MWCO ϭ 10,000). Metal content analyses of the enzyme before and after the anaerobic cyanide treatment in the presence of excess Q 1 revealed that a substantial amount (30%) of copper ions was released in the filtrate after the treatment (Table II). These observations suggest that the 2169 cm Ϫ1 band arose from a low molecular weight copper-cyano species (i.e. a Cu B ⅐CN complex) released from bo-type ubiquinol oxidase. EPR analysis revealed no indication of a Cu 2ϩ ion in the enzyme preparation that showed the 2169 cm Ϫ1 band, suggesting that this Cu B ⅐CN complex was in reduced state (i.e. Cu 1ϩ state).
There are several reports describing the release of copper ions from copper proteins in the presence of cyanide. For cytochrome c oxidase both the Cu A and Cu B centers of the airoxidized enzyme could be removed by dialysis against a CNcontaining solution (41,42). It must be noted, however, that the conditions are very different from the one in the present study. The dialysis was done at alkaline pH (i.e. pH 10), and a much higher concentration (50 mM ϳ1.0 M) of cyanide was required (41,42). Among the previous reports, our particular interest is the cyanide binding study for Cu/Zn-superoxide dismutase from bovine erythrocyte (43). Raman and FT-IR spectroscopic studies revealed that the native Cu 2ϩ -superoxide dismutase binds one cyanide showing a band at 2137 cm Ϫ1 . With increased concentration of cyanide the 2137 cm Ϫ1 band became weaker, and strong vibrational modes (2123, 2093, and 2075 cm Ϫ1 ) developed concomitantly. These bands were due to di-, tri-, and tetracyano Cu 1ϩ complexes, respectively, arising from copper removed from the protein. Simultaneously a new band appeared at 2169 cm Ϫ1 having an abnormally large 13 CN shift of Ϫ60 cm Ϫ1 (43). This 2169 cm Ϫ1 species was also observed in the filtrate through a membrane filter (43). All of these observations strongly suggest that the 2169 cm Ϫ1 species found for the superoxide dismutase-CN system is identical with the 2169 cm Ϫ1 species in the present study. Han et al. (43) concluded that the 2169 cm Ϫ1 species was neither a protein species nor a microcell of CuCN solid because of its abnormal cyanide isotopic shifts.
The 8 cm Ϫ1 D 2 O downshift observed for the 2169 cm Ϫ1 species is not likely due to a direct hydrogen bonding of an H 2 O (or D 2 O) molecule in medium to the CN moiety of the Cu 1ϩ ⅐CN complex, as hydrogen bonding becomes weakened by an H-D exchange (44). For the horseradish peroxidase (Fe 2ϩ )-CN system, an 8 cm Ϫ1 D 2 O upshift (from 2029 to 2037 cm Ϫ1 ) of the C-N stretching frequency has been attributed to the formation of a hydrogen bond between the heme-coordinated cyanide anion and a protonated (or deuterated) distal His residue (37). It is more likely, therefore, that an H 2 O (or D 2 O) molecule itself also participates in forming the Cu B 1ϩ ⅐CN complex and the resulting intramolecular interactions between cyanide(s) and a water ligand(s) may be essential for the appearance of the 2169 cm Ϫ1 band and its unusual vibrational mode pattern.
We have tried to prepare the Cu 1ϩ ⅐CN complex by mixing copper(I) chloride and/or copper(I) cyanide with varying amounts of cyanide in aqueous solution, but without success. It was reported that a reaction involving copper(I), cyanide, and The wild-type (0.51 mM) and the bound Q 8 -free (0.21 mM) oxidases in the air-oxidized state were incubated with 5 mM 12 C 14 N under N 2 atmosphere at 4°C in the dark (wild-type/CN and bound Q 8 -free/CN, respectively). The wild-type ubiquinol oxidase contains about 2.2 mol of Q 8 /mol of the enzyme. A part of the wild-type/CN sample was partially reduced with the ␤-NADH and phenazine methosulfate system as described in the text, and incubated (wild-type/CN/RD). Q 1 was added to a part of the bound Q 8 -free/CN sample at a final concentration of 1 mM and incubated under the same conditions (bound Q 8 -free/Q 1 /CN). After the incubation of each sample was washed with 50 mM Tris-HCl buffer (pH 8.0) containing 0.1% sucrose monolaurate on the membrane filter (MWCO ϭ 10,000). The sample was then diluted with the same buffer and 0.10 mM enzymes were subjected to the metal analysis. water under conditions of high temperature and pressure gave a by-product species with (Cu 3 (CN) 3 (H 2 O)) n structure, comprising a two-dimensional polymer (45). We propose that this kind of species may be responsible for the 2169 cm Ϫ1 band.
Mechanism of the Formation of the Cu B 1ϩ ⅐CN Complex-The partially reduced conditions seem essential for the formation and release of the Cu B 1ϩ ⅐CN complex. Indeed the 2169 cm Ϫ1 species could be quickly produced by anaerobic partial reduction of the CN-inhibited enzyme but not at all from the fully reduced form. The absence of the 2034.5 cm Ϫ1 band that is characteristic to the Fe O 2ϩ -CϭN adduct may be noticed when the 2169 cm Ϫ1 species developed fully by the autoreduction of the CN-inhibited enzyme. This observation is consistent with the notion that the presence of the Cu B center is essential for the binding of cyanide to the reduced enzyme as discussed in the previous section.
The CN-inhibited bound Q 8 -free oxidase resulted in neither the development of the 2169 cm Ϫ1 band nor the release of the Cu B center. A simultaneous addition of excess Q 1 with cyanide to the bound Q 8 -free oxidase was not so effective. These observations suggest that the precise structure around the binuclear site or the quinone binding site(s) may be essential for the formation of the Cu B ⅐CN complex. The role of the loosely bound ubiquinones (Q 8 or Q 1 ) is not clear, but is likely only for providing electron equivalents to the metal centers of ubiquinol oxidase by an unknown mechanism. This is based on the observation of the 2169 cm Ϫ1 species in the one-fourth-reduced or one-half-reduced conditions of the PEG-treated CN-inhibited ubiquinol oxidase, which contains only one molecule of Q 8 at the high affinity binding site.
It is of great importance that the same 2169 cm Ϫ1 species could be produced by anaerobic partial reduction of the CNinhibited cytochrome c oxidase in which no ubiquinone molecule is bound. This observation suggests that there is a common intermediate structure at the binuclear center of the hemecopper respiratory oxidases in the partially reduced CN-inhibited state which is very susceptible to the cyanide binding(s) and release of the Cu B center. The greater formation of the 2169 cm Ϫ1 species for bo-type ubiquinol oxidase than cytochrome c oxidase may be related to the instability of the Fe O 3ϩ -CϭN species (although its presence was confirmed with the g z ϭ 3.24 EPR signal) in the partially reduced CN-inhibited enzyme. This difference is also likely due to a slight difference(s) of the cyanide coordination structure (including distal His residues) at the binuclear center. The unique property of the heme-copper oxidase revealed in the present study may provide a clue for understanding a mechanism of the dioxygen reduction chemistry and the redox-linked proton pumping.