Simultaneous resonance Raman detection of the heme a3-Fe-CO and CuB-CO species in CO-bound ba3-cytochrome c oxidase from Thermus thermophilus. Evidence for a charge transfer CuB-CO transition.

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.

Cytochromes ba 3 and caa 3 serve as the terminal heme-copper oxidases in the Gram-negative thermophilic eubacterium Thermus thermophilus (1). Both enzymes couple the reduction of dioxygen to proton translocation across the inner bacterial membrane, and in contrast to the eucaryotic heme-copper oxidases, catalyze the reduction of NO to N 2 O (1, 2). Cytochrome ba 3 contains a homodinuclear copper complex (Cu A ), one low spin heme b, and a binuclear center that consists of Cu B and heme a 3 (1). The comparative kinetics data on CO photodissociation and rebinding of various heme-copper oxidases and the derived activation parameters have indicated that the COligation/release mechanism in cytochrome ba 3 follows that found in other heme-copper oxidases (3)(4)(5)(6)(7)(8)(9) and proceeds according to the following scheme.
Fe 2ϩ -CO, Cu B ϩ A B SCHEME 1 In contrast to the bovine aa 3 oxidase, Cu B of cytochrome ba 3 has a relative high affinity for CO (k 1 Ͼ 10 4 ), whereas the transfer of CO to heme a 3 2ϩ is characterized by a small k 2 ϭ 8 s Ϫ1 and by a k Ϫ2 ϭ 0.8 s Ϫ1 that is 30-fold greater than that of the bovine aa 3 (3,4).
The FTIR 1 and time-resolved FTIR studies of the CO-bound fully reduced form of ba 3 have revealed several unique characteristics of the enzyme including the formation of the equilibrium Cu B -CO complex and the identification of a ligand docking site (4,6,7). It was reported that the rate of decay of the transient Cu B -CO complex that is produced from the photolysis of complex B (Scheme 1) is 34. 5 s Ϫ1 and rebinding to heme a 3 occurs with k 2 ϭ 28.6 s Ϫ1 (4). The (CO) of the transient Cu B -CO species at 2053 cm Ϫ1 is the same as that of the equilibrium Cu B -CO species and remained unchanged in the pH and pD 5.5-10 ranges, indicating that no structural change takes place at Cu B between these states (4). Recently, the role of the ring A propionate of heme a 3 -Asp 372 -H 2 O site as a proton carrier to the exit/output proton channel (H 2 O pool) that is conserved among all structurally known heme-copper oxidases and is part of the Q-proton pathway in ba 3 -cytochrome c oxidase was reported (8). Further FTIR studies provided solid evidence that in cytochrome ba 3 the ligand delivery channel is located at the Cu B site, which is the ligand entry to the heme a 3 pocket (5). It was suggested that the properties of the O 2 channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba 3 with O 2 and NO. Therefore, it is of particular interest to gain insight into the environment of the spectroscopic silent Cu B .
Resonance Raman (RR) is a structure-sensitive technique, and thus, data of the CO-bound adducts of ba 3 can be interpreted to yield specific information concerning heme/Cu B geometric properties and heme/Cu B -axial ligand bonding interactions (10 -17). Additional information concerning the two different conformations of the heme-copper oxidases, termed ␣ and ␤, have also been provided by FTIR and RR studies (10 -19). Although the presence of one or the other conformation has been implicated in the enzymatic activity of heme-copper oxidases, their functional significance remains to be determined (14). In the present work, we have studied the CO-bound ba 3 oxidase by RR with the aim to fully characterize complexes A and B and thus to explore the proposed role of Cu B in the formation of either the ␣or the ␤-conformation and to determine the significance of the ␣and ␤-conformations in the enzymatic activity of heme-copper oxidases. Our data demonstrate, despite the extreme values of the Fe-CO and C-O stretching vibrations at 507 and 1973 cm Ϫ1 , respectively, the presence of the ␣-conformation in the catalytic site of the enzyme. The analysis of the data also reveals that the environment of Cu B does not control the strength of the Fe-C bond and thus the type of conformation of the binuclear center. The results presented here and those of caa 3 , in contrast to previous reports, demonstrate that enzymatic activity is not controlled by the type of conformation (␣ or ␤) of the binuclear center. We have also made the novel RR characterization of the Cu B -CO complex by its CO stretching frequency at 2053 cm Ϫ1 . The 2053 cm Ϫ1 band, as opposed to 507 and 1973 cm Ϫ1 bands, is more readily observed with 413.1 nm RR excitation than with 428.7 nm excitation, indicating the presence of a metal-to-ligand charge transfer (MLCT) Cu B -CO transition state near 410 nm. This observation provides for the first time an indication as to how to exploit the environment of the spectroscopic silent Cu B center that has been implicated not only in the catalytic properties of heme-copper oxidases but also in the proton pumping mechanisms.

EXPERIMENTAL PROCEDURES
Cytochrome ba 3 from T. thermophilus was isolated by the procedure published previously (1, 4 -6). Resonance Raman spectra were obtained from 40 -50 M samples, pH 7.5, in a cylindrical quartz spinning cell. The RR data were acquired as described elsewhere (10,11,13). FTIR spectra were recorded from 400 M samples at 4 cm Ϫ1 resolution with a Bruker Equinox 55 FTIR spectrometer equipped with a liquid nitrogen cooled mercury cadmium telluride detector. The fully reduced CO samples were anaerobically loaded into a cell with CaF 2 windows and a 0.025-mm spacer. An average of 2000 scans was used for each spectrum. Fig. 1 shows the low frequency RR spectra of CO-bound ba 3 at neutral pH obtained with 428.7 nm excitation, in which resonances from heme b 2ϩ and heme a 3 -CO are enhanced (4). We detect two modes that have carbon and oxygen isotopic sensitivity. The shifts are more evident in the difference spectrum (trace c) between the 12 C 16 O-and 13 C 18 O-bound states. The (Fe-CO) at 507 cm Ϫ1 is 12-15 cm Ϫ1 lower than those of the aa 3 -type oxidases from P. denitificans (10), aa 3 -600 (11), bovine heart (12,13), and Rhodobacter sphaeroides (14,15). In addition, the shift of the weak mode at 568 cm Ϫ1 (trace a) to 550 cm Ϫ1 (trace b) also appears in the isotope difference spectrum (trace c). This mode is attributed to the Fe-C-O bending mode and is 5-10 cm Ϫ1 lower than that of the aa 3 -type oxidases (10 -15).

RESULTS
In Fig. 2A we present the high frequency region of the RR spectra. The difference spectrum (trace a) between 12 Fig. 2B shows that the band pair 2053/1959 cm Ϫ1 is more enhanced with 413.1 nm excitation. The opposite occurs for the 1973/1884 cm Ϫ1 band pair. It is readily observed with 428.7 nm excitation, but it has lost most of its intensity in the 413.1 nm excitation spectra. This is consistent with the optical absorption spectrum of the CO-bound heme a 3 at max ϭ 430 nm (4). We attribute the increased intensity of the 2053/1959 cm Ϫ1 band pair to the presence of a Cu B -CO MLCT transition near 410 nm. We have been unable, however, to detect it by absorption spectroscopy because it is obscured by the strong Soret transition. Our assignment finds support from the absorption spectrum of the CO bound form of copper/topa quinone-containing amine oxidase from Arthrobactor globiformis where two transitions with maximum absorbance at 334 and 434 nm were observed (20). The excitation laser wavelength at 428.7 nm was obtained from a diode laser (Melles Griot). The accumulation time was 180 min for each spectrum and the laser power was 50 W. Inset, correlation between frequencies of the Fe-CO versus C-O stretching modes for the ␣and ␤-forms of heme-copper oxidases. ࡗ, cytochrome ba 3 from T. thermophilus; , cytochrome aa 3 from P. denitrificans (10); f, cytochrome aa 3 -600 from B. subtilis (11) and mammalian cytochrome c oxidase (12,13); Š, the ␣-form of cytochrome aa 3 from R. sphaeroides (14,15); q, cytochrome bo 3 from Escherichia coli (16); OE, cytochrome cbb 3 from Rhodobacter capsulatus (17); ‹, the ␤-form of cytochrome aa 3 from R. sphaeroides (14,15); छ, myoglobins and hemoglobins (14,15,17).

The Heme a 3 -Cu B Moiety of ba 3 Cytochrome c Oxidase 22792
the lowest and highest, respectively, ever reported among the heme a 3 -containing heme-copper oxidases. Nevertheless, the (Fe-CO) and (CO) frequencies fall on the correlation curve for the ␣-form of the enzyme and suggest that it has the same structure as that of the major form of the aa 3 -type oxidases (10,13,15). It has been proposed that the enhancement of the bending mode in the ␣-form is the result of steric forces which caused it to be bent or tilted and that Cu B serves to lower its symmetry so that ␦(Fe-C-O) is enhanced (14,15). A high I ␦ /I (intensity of bending mode/intensity of stretching mode) is an indication of a strong interaction between the CO and Cu B (10,16,17). Although the frequencies we detect in ba 3 are quite different from those reported for the aa 3 -type oxidases, the I ␦ /I (0.3) is very similar. Thus, Cu B exerts similar steric forces to Fe-C-O in the ba 3 -and aa 3 -type oxidases. The anomalously high frequency of the Fe-CO stretching mode at 517-524 cm Ϫ1 in the ␣-form of the aa 3 -and bo 3 -type oxidases has also been attributed to distal effects exerted by Cu B (12, 14 -16). The frequency of the (Fe-CO) in ba 3 is close to those of model heme and heme proteins and of the ␤-form of heme-copper oxidases in which CO can bind to the heme Fe without anomalous polar or steric interactions (14 -16). Based on the above arguments, the low frequency of (Fe-CO) in ba 3 cannot be attributed only to distal effects caused by Cu B to the bound CO. It is known that (Fe-CO) and (CO) reflect both the identity and properties of the proximal ligand because the bound CO competes with the proximal ligand for the same d z 2 orbital and the polarity of the distal heme pocket (10). A highly polar environment favors -back donation, resulting in an increased (Fe-CO) and reduced (CO) due to the increased density in the CO antibonding orbitals. Taken together, the present results and those recently reported for the aa 3 -type oxidases indicate that Cu B contributes to distort the Fe-C-O group from its axial symmetry so that the bending vibration is enhanced but does not affect the strength of the Fe-CO bond. In the absence of polar interactions, we suggest that the strength of the proximal histidine (His 384 ) H-bonding interaction affects the strength of the Fe-C and C-O bonds. This explanation finds support from the crystal structure of ba 3 where it has been shown that the distance from N ␦ of His 384 to the carbonyl of Gly 359 is 3.43 Å (H-bonding distance).
In the past, it has been suggested that the major conformations (␣ and ␤) at the binuclear center and the existence of equilibrium between them might have significant importance in the enzymatic activity of heme-copper oxidases (14,15,17,18). For instance, in certain mutant heme-copper oxidases, low frequency Fe-CO stretching modes were detected (␤-form), and the enzyme had no dioxygen reduction activity (19). However, due to the presence of both forms the activity of the population with the ␤-form could not be determined. The ␣and ␤-conformational states of heme-copper oxidases, although their functional significance and the origin for the splitting have not been established, have been attributed to changes in the distance between the iron atom of heme a 3 and Cu B (10,14,15). It was postulated that the different structures result from a change in the position of the Cu B atom with respect to the Fe-CO due to the presence of one or more ionizable groups (14,15). Possible candidates included the cross-linked, conserved tyrosine that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu B . We have recently demonstrated that that the absence of the farnesyl-tyrosine linkage has no direct control on the (CO) stretching frequency and thus on the type (␣ or ␤) of the stable conformation that is present in heme-copper oxidases (4,5,9,21). It has also been established that the Cu B -His environment is very rigid and not subject to conformational transitions that are associated with protonation/deprotonation events of the Cu B His ligands (5). The binuclear centers of ba 3 (␣-form) and caa 3 (␤-form) oxidases contain the conserved tyrosine; therefore, we can exclude that the crosslinked tyrosine provides the ionizable group that causes the heme-Cu B distance to change. In the absence of any evidence regarding the involvement of functional groups associated within the binuclear center, we suggest that the presence of one or more ionizable groups near the binuclear center that affect the heme a 3 -CO Cu B distance are the cause of the observed conformations observed in aa 3 oxidases. Given that the binuclear centers in ba 3 and its counterpart caa 3 oxidase are very similar, the same argument holds for the absence of the ␤-form in ba 3 oxidase but its presence in its counterpart caa 3 . Taken together, the present data and data previously reported (4,5,9,21) demonstrate that fully active heme-copper oxidases can have binuclear centers in which either the ␣or the ␤-form is present.
The Cu B -CO Complex-The variation of (CO) in mononuclear Cu-containing proteins has been attributed to changes in the coordination structure of the metal (20). The insensitivity of the (CO) of Cu B frequency to H 2 O/D 2 O exchange and to pH/pD 5.5-9.7 range indicated that the degree of back-donation of electron density from the d orbitals to the antibonding * orbitals is not altered under these conditions (5,8). In the presence of small amounts of O 2 , however, the (C-O) of Cu B 1ϩ -CO at 2053 cm Ϫ1 (complex A) shifts to 2045 cm Ϫ1 and remains unchanged in H 2 O/D 2 O exchanges and in the pH 6.5-9.0 range (5). This observation was attributed to an increased electron density in the CO antibonding orbitals that results in weakening the C-O bond strength (lower CO ). It was concluded that the change in the (CO) of complex A results in an increase of k Ϫ2 and thus to a higher affinity of Cu B for exogenous ligands. The nature of ligands coordinated to the metal in copper proteins has been of particular interest but difficult to determine, since conformational and/or chemical changes in the proteins may lead to ligand exchanges by the EPR silent Although we have identified additional vibrations in the RR spectra (data not shown) that can be attributed to the internal vibrations of Cu B -(N-His), the insufficient amounts, so far, of 15 N-enriched enzyme have prevented us from collecting high quality RR data of the 15 N-enriched enzyme. Therefore, it remains to be established whether internal ligand motions are resonance-enhanced, either because the ligand coordinates themselves are displaced upon MLCT excitation or the ligand coordinates contribute to ground-state normal modes that involve Cu B -L(N) stretching coordinates. In summary, the unique CO binding properties of ba 3 oxidase have allowed us, first, to investigate from the frequencies and intensities of Fe-C-O the role of Cu B in the structure of this coordinated ligand. We suggest that Cu B maintains the same role in the structure of the physiological coordinated ligands, such as NO and O 2 . This way, the role of Cu B in the structure-function relationship has been established. Second, we have identified the Cu B -CO complex by its (CO) at 2053 cm Ϫ1 . The (CO) is strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. The RR identification of the Cu B -CO complex provides us direct access, for the first time, to spectroscopic silent Cu B site under steady state conditions. This way, the environment of Cu B that has been implicated in both the O 2 delivery channel and proton translocation mechanisms can now be explored (5,8).