Cytochrome oxidase (a3) heme and copper observed by low-temperature Fourier transform infrared spectroscopy of the CO complex.

Abstract

Carbon monoxide bound to iron or copper in substrate-reduced mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) from beef heart has been used to explore the structural interaction of the a3 heme-copper pocket at 15 K and 80 K in the dark and in the presence of visible light. The vibrational absorptions of CO measured by a Fourier transform infrared interferometer occur in the dark at 1963 cm-1, with small absorptions near 1952 cm-1, and are due to a3 heme--CO complexes. These disappear in strong visible light and are replaced by a major absorption at 2062 cm-1 and a minor one at 2043 cm-1 due to Cu--CO. Relaxation in the dark is rapid and quantitative at 210 K, but becomes negligible below 140 K. The multiple absorptions indicate structural heterogeneity of cytochrome oxidase in mitochondria. The Cu--CO absorptions (vCO) are similar to those in hemocyanin--CO complexes from molluscs (vCO - 2062 cm-1) and crustaceans (vCO = 2043 cm-1). The 2062 cm-1 Cu--CO absorption of cytochrome oxidase is split into two bands at 15 K. Analysis of spectral data suggest the presence of a very nonpolar heme--Cu pocket in which the heme-CO complex is highly ordered, but in which the Cu--CO complex is much more flexible, especially above 80 K. A function for these structures in oxygen reduction is proposed.

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.…”

“…2 (traces A-G) the frequency of the CO mode associated with Cu B is invariant between pD 5.5 and 9.7 and unaffected by H/D exchange, consistent with no change in the protonation of groups in close proximity to CO under our experimental conditions. The frequency of (CO) that we observe is similar to those of Limulus hemocyanin ( CO ϭ 2053 cm Ϫ1 ) (27) and nitrite reductase from Alcagenes faecalis ( CO ϭ 2050 cm Ϫ1 ) (30) but significantly different from that found in other Cu B -containing oxidases (14,21,23,26,31,32). In the absence of steric constraints, CO binds to Cu B in a linear fashion (M-C-O), as required for optimal bonding between the d orbitals of Cu and the antibonding * of CO. Back-donation of electron density from the d orbitals to the antibonding * orbitals strengthens the Cu-C bond and weakens the C-O bond.…”

“…2, traces A-B and B-G). The ratio of the relative areas of the Fe-CO/Cu-CO is 4:1 in the absolute spectra, and considering the integrated absorptivities expected of CO complexes of heme and copper proteins (26), the ratio of ⑀ Fe-CO /⑀ Cu-CO is ϳ1.5:1 suggesting ϳ30% of unligated heme a 3 , in agreement with the magnetic circular dichroism (17) and optical data (19). 13 CO; E, pD 6.5; F, pD 8.5; and G, pD 9.7.…”

Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System

“…The role has never been proposed until this study, although CO binding to Cu B was discovered by FTIR analysis 32 years ago (37).…”

Bound water in the proton translocation mechanism of the haem‐copper oxidases

“…<----trough and peak at 1960 cm -1 and 2065 cm -I show the wellknown disappearance of the C---O bound to haem iron, and the appearance of C-= O bound to Curs, respectively [21,22]. We have recently shown that this perturbation shifts the carboxylic acid infrared C = O stretch of Glu 212 in the boa enzyme (where it is Glu 2s~) near 1730 cm 1 suggesting connectivity between a CuB histidine ligand and the glutamic acid [18].…”