Photoperturbation of the heme a3-CuB binuclear center of cytochrome c oxidase CO complex observed by Fourier transform infrared spectroscopy

Abstract

Purified cytochrome c oxidase CO complex from beef heart has been studied by Fourier transform infrared absorbance difference spectroscopy. Photolysis at 10-20 Kelvin results in dissociation of a3FeCO, formation of CuBCO, and perturbation of the a3-heme and CuB complex. The vibrational perturbation spectrum between 900 and 1700 cm-1 contains a wealth of information about the binuclear center. Appearance in infrared photoperturbation difference spectra of virtually all bands previously reported from resonance Raman spectra indicate the importance of polarization along the 4-vinyl:8-formyl axis, which results in the reduction of heme symmetry to C2v. Frequency-shifted bands due to the 8-formyl and 4-vinyl groups of the a3-heme have been identified and quantitated. The frequency shifts have been interpreted as being due to a change in porphyrin polarization with change in spin state of the iron by photodissociation of CO or perturbation of the CuB coordination complex.

Observation of the Equilibrium CuB-CO Complex and Functional Implications of the Transient Hemea3 Propionates in Cytochromeba3-CO from Thermus thermophilus

“…In the ␤-form, the frequencies are placed on the correlation curve (22), and in the ␥-conformation, Cu B is moved closer to the CO-bound heme a 3 , thereby the Fe-C-O moiety is further distorted from its preferred symmetry in the ␣-form (28). The frequency of the CO mode of the Cu B 1ϩ -CO complex at 2053 cm Ϫ1 , however, is 10 -12 cm Ϫ1 higher and 8 -12 cm Ϫ1 lower than the corresponding frequencies of the ␤-and ␣-forms found in other hemecopper oxidases (23,25), and thus, we are unable to assign it to either the ␣-or the ␤-form at present. Although the species we detect in the FTIR data could arise from a heme a 3 -CO/Cu B 1ϩ -CO complex, consideration of k Ϫ2 (0.8 s Ϫ1 ) suggests that the peaks we detect are associated with the formation of complexes A and B (Scheme 1).…”

“…We assign the peaks at 1967, 1973, and 1982 cm Ϫ1 to the C-O stretching modes of heme a 3 -CO (complex B), and the peak at 2053 cm Ϫ1 to the C-O stretching mode of Cu B 1ϩ -CO (complex A). The mode at 1967 cm Ϫ1 is similar to that found for the ␣-form in the CO adducts of aa 3 oxidases from bovine heart, Rhodobacter sphaeroides, aa 3 -600, and P. denitrificans (21)(22)(23)(24)(25)(26)(27)(28), and the 1973 cm Ϫ1 mode is 2 cm Ϫ1 lower than the ␥-form found in P. denitrificans (28). The frequency of the 1982 cm Ϫ1 mode, however, does not coincide with the ␤-form found in other heme-copper oxidases (20 -23), and thus, we suggest that it represents a structure of the active site in which Cu B exerts a strong steric effect on the heme a 3 -bound CO.…”

“…A, step-scan TR-FTIR difference spectra of the CO-bound form of fully reduced cytochrome ba 3 (pD 8.5) at 0.15, 0.5, 1, 2, 3, 4, 5,6,7,8,9,10,15,20,21,22,23,25,30,35,40,45,50,55,60,64,70, and 78.5 ms subsequent to CO photolysis. The spectral resolution was 8 cm Ϫ1 , and the time resolution was 100 s. A 532 nm, 3-10 mJ/pulse pump beam was used for photolysis.…”

Fourier Transform Infrared (FTIR) and Step-scan Time-resolved FTIR Spectroscopies Reveal a Unique Active Site in Cytochromecaa3 Oxidase from Thermus thermophilus

“…The frequency of the major CO mode in cytochrome caa 3 is 8 and 5 cm Ϫ1 lower than the corresponding frequencies found in CO-bound heme a 3 from P. denitrificans and mammalian, respectively (8,10,15). It is very similar, however, to that found in cbb 3 oxidase whose binuclear center does not consist of o 3 -or a 3 -type heme (14).…”

“…The Fourier transform infrared (FTIR) spectra of the CObound heme-copper oxidases have revealed several characteristics of the binuclear pocket, including, from the frequencies of the C-O stretching modes of heme iron and Cu B , the identity of the metal to which the CO is bound, as well as the interactions between the axial ligands and the heme and/or the Cu B environment (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Cryogenic techniques have also been used successfully in conjunction with FTIR to cryotrap metastable intermediates by first freezing the CO-bound protein at cryogenic temperatures and then photodissociating the CO (6 -10).…”