Observation of the Fe4+ = O stretching Raman band for cytochrome oxidase compound B at ambient temperature.

Resonance Raman and visible absorption spectra were simultaneously observed for cytochrome oxidase reaction intermediates at 5 degrees C by using the artificial cardiovascular system (Ogura, T., Yoshikawa, S., and Kitagawa, T. (1989) Biochemistry 28, 8022-8027) and a device for Raman/absorption simultaneous measurements (Ogura, T., and Kitagawa, T. (1988) Rev. Sci. Instrum. 59, 1316-1320). The Fe4+ = O stretching (nu FeO) Raman band was observed at 788 cm-1 for compound B for the first time. This band showed the 16O/18O isotopic frequency shift (delta nu FeO) by 40 cm-1, in agreement with that for horseradish peroxidase compound II (nu FeO = 787 cm-1 and delta nu FeO = 34 cm-1). In the time region when the FeII-O2 stretching band for compound A and the nu FeO band for compound B were coexistent, a Raman band assignable to the Fe3+-O-O-Cu2+ linkage was not recognized.


Resonance
Raman and visible absorption spectra were simultaneously observed for cytochrome oxidase reaction intermediates at 5 "C by using the artificial cardiovascular system (Ogura, T., Yoshikawa with that for horseradish peroxidase compound II (vF~~ = 787 cm-' and AVF~~ = 34 cm-'). In the time region when the Fe"-O2 stretching band for compound A and the VFeO band for compound B were coexistent, a Raman band assignable to the Fe3+-O-O-Cu2+ linkage was not recognized.
Cytochrome oxidase (cytochrome c:oxygen oxidoreductase, EC 1.9.3.1), the terminal enzyme of the mitochondrial respiratory chain, catalyzes reduction of molecular oxygen to water and at the same time couples the reaction with proton translocation across the energy-transducing membrane (1,2). The electrochemical potential thus generated is utilized to phosphorylate ADP. Mammalian cytochrome oxidases contain two heme a groups called cytochromes a and a3 and two copper ions named CuA and CUE. Cytochrome a/C& moiety receives electrons from cytochrome c and transfers them to the cytochrome a&us binuclear center. Dioxygen binds to the Fe(I1) ion of cytochrome a3 and is converted to water when four electrons are supplied. The mechanism of dioxygen reduction has been mainly investigated with visible absorption (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and EPR (15)(16)(17)(18) spectroscopies, but these methods hardly provide definite structural information on bound oxygen. Resonance Raman (RR)' spectroscopy is a powerful technique for studying a structure of heme and its vicinity (19)(20)(21) and indeed provided important information on cytochrome oxidase (22)(23)(24)(25)(26)(27)(28). Particularly the RR spectra of transient species (25)(26)(27)(28) seemed to bring about essential data on structures of intermediates, but observation of RR spectra with a much higher signal-to-noise ratio is required for examination of those labile intermediates. Since the reaction intermediates of cytochrome oxidase have been defined on the basis of visible absorption spectra, it is extremely desirous to correlate the observed RR spectra with visible absorption spectra. Accordingly, we constructed a device for Raman/ absorption simultaneous measurements (29) and also a sample flow system (artificial cardiovascular system) (30), with which the reacted enzyme is automatically regenerated during circulation, in order to accumulate the RR spectra of intermediates for a long time with a limited amount of the enzyme. Combination of these two apparatus enabled us to observe the Fe(II)-O2 stretching RR band for compound A (31) whose frequency was in agreement with the corrected value of Varotsis et al. (28)   A, compound A reproduced from Ref. 31 (path length = 1 mm); B, spectrum 1A -2.5. spectrum 24. The subtraction factor of 2.5 was chosen so as to minimize the contribution from compound A at 586 nm to the resultant spectrum. The M in the figure corresponds to 0.007 and 0.01 for spectra A and B, respectively, but since the path lengths are different and the enzyme concentration might be slightly different, comparison of relative absorbances between the two spectra needs some corrections. served simultaneously.
The absolute spectrum indicated about 90% of carbon monoxide was photodissociated. These two spectra are quite alike and resemble the difference spectrum of compound B uersus the fully reduced form (8). However, these spectra appeared to involve some extent of contribution from compound A. This is proved by the calculation of difference spectra explained below.
Spectrum A in Fig. 2 represents the difference spectrum of compound A with regard to the CO-photodissociated species, which was obtained in the previous study (31) with the same apparatus but with a thinner cell (1 mm) for the shorter residence time of the sample in the light beam (200 rs). Spectrum B in Fig. 2 was obtained by subtracting Fig. 2A from Fig. 1A. The positive contribution at 586 nm implicitly seen in Fig. lA disappeared in Fig. 2B, and the resultant spectrum is practically identical with that of compound B reported previously (30) and also with the 500-100-rs difference spectrum of Orii (14). It is noted that the difference peak at 549 nm is prominent in Fig. 2B but is absent in Fig. 2A, indicating that electrons of cytochrome c are transferred to cytochrome oxidase at the stage of compound B but not at the stage of compound A. Thus, it became evident that both compound A and compound B are coexistent in the volume of light beam under the present laser power and flow speed. It is noted that when the laser power was raised for the same flow speed, the reaction occurred faster, and the spectrum of compound A was not seen.
Since the oxygen-associated Raman bands of oxygen ad-ducts of hemeproteins and metalloporphyrins are expected to appear in the 500-1200 cm-l region, we examined the RR spectra in that wave number region. Fig. 3 displays the RR spectra of the cytochrome oxidase intermediate for reaction with i602 (A) and "02 (B) excited at 418 nm and their difference (C = A -B). Spectra A and B in Fig. 3 were measured simultaneously with the absorption spectra of Fig.  1, A and B, respectively. Although the two RR spectra are similar, their difference spectrum shown by Fig. 3C evidently indicates the presence of two isotope-sensitive bands. The fact that the difference spectrum shows no difference at 683 cm-' where the most intense porphyrin in-plane bands (I+) were observed makes the presence of the oxygen isotopesensitive bands more reliable. The same difference spectra were repeatedly observed for independent preparations of the sample.
The  the previous observation for the Fe(II)-OS stretching vibration of compound A (31) and also with a very recent report for mixed valence oxygenated cytochrome oxidase (32) and that of the corrected values of Varotsis et al. (28). Accordingly, the 569/540 cm-' bands for the *60Z/1sO~ derivatives are assigned to compound A with no doubt. The other oxygen isotope-sensitive band at 788 cm-' for the "OZ derivative, which should be assigned to compound B, exhibits a downshift by 40 cm-' for the "O* replacement. The magnitude of the '60/180 frequency shift is close to the value expected for the Fe0 diatomic oscillator which is calculated to be 35 cm-' in the harmonic approximation. (If Fe were assumed to be firmly bound to the porphyrin core, the expected shift would be 45 cm-'.) So far the vreo bands have been observed around 760-800 cm-' for hemeproteins (34-40) and around 800-850 cm-' for five-and six-coordinate oxoferryl-porphyrin complexes at low temperatures (41-43) as listed in Table I. Judging from these values and their '60/1s0 isotopic frequency shift, it is quite reasonable to assign the 788 cm-' band of compound B to the Fe4+=0 stretching vibration of the six-coordinate heme.
The Oreo frequency of HRP compound II is located at 787 and 775 cm-' for the alkaline and neutral forms, respectively, and the lower frequency of the neutral form was attributed to hydrogen bonding of the bound oxygen to a distal residue (37). In the presence of the hydrogen bond, the bound oxygen was exchanged with that of bulk water, and the "jO/"O isotopic frequency shift was not observable when H*180Z was reacted in Ht160. The vi+o frequency of cytochrome oxidase compound B is closer to that of the alkaline form, and the 160/1'0 isotopic frequency shift was clearly observed in HZ1'jO. This may suggest that the oxygen atom of oxoferryl heme in compound B is not hydrogen-bonded and not exchanged with bulk water. However, the relative intensity of the '60/'80 difference peak of compound B in Fig. 3C compared with that of compound A is not as large as expected from the difference in their lifetimes. This might imply that a part of oxoferryl oxygen was exchanged with water while the measurement was performed. It is emphasized that spectrum C in Fig. 3 exhibits no difference feature between 800 and 1200 cm-' where the -O--O--stretching band would be expected to appear if the Fe3+-0-0-Cu*+ linkage were formed.
In conclusion, this study definitely demonstrated that compound B, a relatively stable intermediate with the absorption spectrum closely similar to that of the fully oxidized form, contains the oxoferryl iron at the dioxygen binding site.