pH-Dependent Structural Changes at the Heme-Copper Binuclear Center of Cytochrome c Oxidase

Abstract

The resonance Raman spectra of the aa3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (alpha and beta) interconvert reversibly in the pH 6-9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D.M. Mitchell, J.P. Sapleigh, A.M.Archer, J.O. Alben, and R.B.Gennis, 1996, Biochemistry 35:9446-9450). It is postulated that the different structures result from a change in the position of the Cu(B) atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu(B) are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.

Simultaneous Resonance Raman Detection of the Heme a3-Fe-CO and CuB-CO Species in CO-bound ba3-Cytochrome c Oxidase from Thermus thermophilus

“…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).…”

“…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).…”

“…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).…”

“…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).…”

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

“…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.…”