The active site structure of ba₃ oxidase from Thermus thermophilus studied by resonance Raman spectroscopy

The ba₃ cytochrome oxidase from Thermus thermophilus was studied by resonance Raman spectroscopy. The component spectra of both heme groups were determined by using different excitation wavelengths. In the ferric state the heme a₃ group reveals resonance Raman marker bands characteristic for two high spin species with the heme iron in an in-plane and an out-of-plane configuration that reflects a coordination equilibrium. This equilibrium obviously results from protonation of one of the axial ligands that is ascribed to a hydroxide. Coordination by its protonated form, a water molecule, may be 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 cm^-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 natural redox partner cytochrome c₅₅₂. These changes of the coordination equilibrium are attributed to the perturbation of the hydrogen bonding network at the catalytic center that is connected to the protein surface via a relay of hydrogen bonds. Environmental changes at the catalytic site are sensitively reflected by the formyl stretching of heme a₃. The unique structural properties of the ba₃ oxidase may be related to the unusual proton pump efficiency and heme a₃ redox potential.