Two conformations of the catalytic site in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Abstract

Resonance Raman spectra of the carbon monoxy derivative of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides show two distinct Fe-CO stretching modes (519 and 493 cm-1) at room temperature. The frequency of the mode at 519 cm-1 coincides with that of other terminal oxidases at neutral pH. Two C-O stretching modes, one at 1966 cm-1 and one at 1955 cm-1, are also found. The splitting of the C-O stretching mode is consistent with the FTIR spectra of cytochrome c oxidases at cryogenic temperatures in which two different conformations (alpha and beta) of the catalytic site of the enzyme are present. The splitting of both the Fe-CO and C-O stretching modes under our conditions indicates that these two forms of the enzyme are also present at room temperature, and with the additional information on the Fe-CO modes provided here, a structural origin for the two forms may be postulated. The alpha-form has the same general structure of the active site as mammalian oxidase, a structure in which the copper atom that is the part of the Fe-CuB binuclear site interacts strongly with the bound CO. We postulate that the copper atom exerts a strong polar or steric effect on the heme-bound CO, resulting in either compression of the Fe-CO bond or distortion of the Fe-CO moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…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.…”

“…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. The ␣-form represents a conformation of the enzyme in which the frequencies of (Fe-CO) and (CO) lie off the correlation curve for heme proteins with a trans-histidine ligand (22). 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 ␣-form represents a conformation of the enzyme in which the frequencies of (Fe-CO) and (CO) lie off the correlation curve for heme proteins with a trans-histidine ligand (22). 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.…”

“…Information concerning the active site of heme-copper oxidases has been deduced from resonance Raman studies of the CO-bound form of the enzymes (22,24,28,34). In one of these studies, Rousseau and co-workers (34), based upon pH dependent changes seen in the heme a 3 Fe-CO stretching frequency, proposed that structural changes that modulate the position of Cu B with respect to the heme-CO are coupled to protonation/ deprotonation events of one or more residues.…”

“…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.…”