Spectroscopic characterization of cytochrome ba3, a terminal oxidase from Thermus thermophilus: comparison of the a3/CuB site to that of bovine cytochrome aa3.
Unliganded and cyano derivatives of cytochrome ba3 from Thermus thermophilus have been examined by UV-vis, EPR, and resonance Raman spectroscopies. Species of cytochrome ba3 investigated include its resting, as-isolated, fully oxidized state, the fully reduced, unliganded enzyme, the one-electron-reduced cyano complex, the three-electron-reduced cyano complex, and the fully reduced cyano complex. Results are compared to those obtained from similar adducts of bovine cytochrome aa3, in particular, the fully reduced cyano complex. Our objective was to identify structural similarities and differences at the ligand-binding binuclear site of the two enzymes. We observed that the inner core skeletal vibrations of cytochrome a3 are the same for similar adducts of the bacterial ba3 and mammalian aa3, indicating similar spin and iron-porphyrin coordination properties resulting in comparable porphyrin core geometries. On the other hand, many of the vibrational frequencies associated with the formyl and vinyl peripheral substituents, and the outer pyrrole carbon atoms differ between the bovine and bacterial enzymes. Use of 57Fe labeled ba3 allows identification of two separate vFe-N(His) frequencies displayed by the fully reduced, unliganded cytochrome. These frequencies, occurring at 193 and 209 cm-1, are ascribed to distinct protein conformers, which are best evidenced by the Fe-N(His) vibrations. This result is again in contrast to the bovine enzyme which has been shown by others to display a single Fe-N(His) stretching frequency at 214 cm-1. The low-frequency Fea3(2+)-CN- vibrations of the three-electron and fully reduced cyano complexes of cytochrome ba3 are identified by using 15N and 13C isotopomers of CN-. These spectral signatures are identical to those reported earlier for the one-electron-reduced cyanide adduct (cytochrome a3 reduced), showing that the Fea3(2+)-CN- vibrational frequencies are independent of the redox states of the other three metal centers. Similarly, the CuB2+ EPR signatures appear similar in both the one-electron- and three-electron-reduced cyanide adducts. On the other hand, the electronic absorption spectra of ferrous alpha 3-CN- show systematic red-shifts of the alpha band as each of the other metal centers is reduced, and other, more subtle, differences in the electronic absorptions of the three-electron-reduced and four-electron-reduced cyanide adducts are revealed in the difference spectra. The relevance of these findings toward explaining the different cyanide binding and redox chemistry described herein and toward establishing the extent of structural analogy between the oxygen binding sites of the two proteins is discussed.