The active site structure of ba(3) oxidase from Thermus thermophilus studied by resonance Raman spectroscopy

The ba(3) cytochrome oxidase from Thermus thermophilus was studied by reson ance Raman spectroscopy. The component spectra of both heme groups were det ermined by using different excitation wavelengths. In the ferric state the heme a, group reveals resonance Raman marker bands characteristic for two h igh spin species with the heme iron in an in-plane and an out-of-plane conf iguration that reflects a coordination equilibrium. This equilibrium obviou sly results from protonation of one of the axial ligands that is ascribed t o a hydroxide. Coordination by its protonated form, a water molecule, may b e too weak to keep the heme iron in the porphyrin plane. The corresponding Fe-OH2 stretching mode was attributed to a weak H/D-sensitive band at 464 c m(-1). The coordination equilibrium not only depends on the pH but is also affected by the buffer, the salt concentration, and the binding of the natu ral redox partner cytochrome c(552). These changes of the coordination equi librium are attributed to the perturbation of the hydrogen bonding network at the catalytic center that is connected to the protein surface via a rela y of hydrogen bonds. Environmental changes at the catalytic site are sensit ively reflected by the formyl stretching of heme a(3). The unique structura l properties of the ba(3) oxidase may be related to the unusual proton pump efficiency and heme a(3) redox potential. (C) 1999 John Wiley & Sons, Inc.