George Heibel

Resonance Raman study of cytochrome aa3 from Sulfolobus acidocaldarius.

The single subunit terminal oxidase of Sulfolobus acidocaldarius, cytochrome aa 3, was studied by resonance Raman spectroscopy. Results on the fully oxidized, the fully reduced, and the reduced carbon monoxide complex are reported and compared with those of sucaryotic cytochrome oxidase. It is shown that in both redox states the hemex a and a 3 are in the six‐coordinated low‐spin and six‐coordinated high‐spin configuration, respectively. The resonance Raman spectra reveal far‐reaching similarities of this archaebacterial with mammalian or plant enzymes except for the reduced form of heme a. The formyl substituent of this heme appears above 1 640 cm−1, ruling out significant hydrogen bonding interactions which is in sharp contrast to beef heart cytochrome oxidase. In addition, frequency upshifts of the marker bands ν4 and ν2 are noted indicating differences in the electron density distribution within the molecular orbitals of the porphyrin.

Structural studies of cytochrome c-554 from Chloroflexus aurantiacus by resonance Raman spectroscopic techniques

The four-heme cytochrome c -554 of Chloroflexus aurantiacus was studied by resonance Raman and surface-enhanced resonance Raman spectroscopy. The resonance Raman spectra show the characteristic vibrational signature of c -type hemes as demonstrated by a comparison with those of horse-heart cytochrome c , which were obtained under identical conditions. Spectral differences between both cytochromes can be related to the porphyrin geometry and the interactions of the heme with the immediate protein environment. Asymmetry of bands of cytochrome c -554 in all spectral regions provides support for conformational heterogeneity among the four hemes. The core-size-sensitive marker bands are interpreted in terms of a slight core contraction of the tetrapyrrole macrocycle in the six-coordinated low-spin states with respect to horse-heart cytochrome c . Shoulders on the low-frequency side of these modes indicate that at least one of the cytochrome c -554 hemes partially exists in a five-coordinated high-spin configuration. The analysis of the low frequency region provides evidence that some of the hemes are located in a more open and flexible protein pocket than in cytochrome c , accounting for the low-lying redox potentials. These structural data are complemented by the surface-enhanced resonance Raman results of cytochrome c -554 adsorbed on colloidal silver. These spectra reveal a significantly larger fraction of the five-coordinated high-spin configuration than in the case of cytochrome c . This can be attributed to a higher susceptibility to conformational distortions of these heme pockets upon electrostatic interactions with the charged silver surface.

Resonance Raman Studies of Bacterial and Mammalian Cytochrome Oxidases

Soret-excited resonance Raman spectroscopy has been used to examine the fully oxidized and fully reduced forms of the cytochrome oxidases from beef heart, the two- and three subunit forms from Paracoccus denitrificans, and the putative quinol oxidase from Sulfolobus acidocaldarius. Spectra are recorded in both the high frequency (1450–1700 cm-1) and low frequency regions (280–460 cm-1). A complete assignment of the high frequency modes for hemes a and a 3 is presented, based on the results of a band fitting procedure, permitting a detailed structural comparison of the bacterial and mammalian enzymes (Heibel et al., 1993a; Heibel et al., 1993b; Hildebrandt et al., 1991).