SPECTROSCOPIC CHARACTERIZATION OF CYTOCHROME BA(3), A TERMINAL OXIDASE FROM THERMUS-THERMOPHILUS - COMPARISON OF THE A(3) CU-B SITE TO THATOF BOVINE CYTOCHROME AA(3)/

Unliganded and cyano derivatives of cytochrome ba(3) from Thermus ther mophilus have been examined by UV-vis, EPR, and resonance Raman spectr oscopies. Species of cytochrome ba(3) investigated include its resting , as-isolated, fully oxidized state, the fully reduced, unliganded enz yme, the one-electron-reduced cyano complex,the three-electron-reduced cyano complex, and the fully reduced cyano complex. Results are compa red to those obtained from similar adducts of bovine cytochrome aa(3), 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 sk eletal vibrations of cytochrome a, are the same for similar adducts of the bacterial ba(3) and mammalian aa(3), indicating similar spin and iron-porphyrin coordination properties resulting in comparable porphyr in core geometries. On the other hand, many of the vibrational frequen cies associated with the formyl and vinyl peripheral substituents, and the outer pyrrole carbon atoms differ between the bovine and bacteria l enzymes. Use of Fe-57 labeled ba(3) allows identification of two sep arate vFe-N(His) frequencies displayed by the fully reduced, unligande d cytochrome. These frequencies, occurring at 193 and 209 cm(-1), are ascribed to distinct protein conformers, which are best evidenced by t he Fe-N(His) vibrations. This result is again in contrast to the bovin e enzyme which has been shown by others to display a single Fe-N(His) stretching frequency at 214 cm(-1). The low-frequency Fe-a3(2+)-CN- vi brations of the three-electron and fully reduced cyano complexes of cy tochrome ba(3) are identified by using N-15 and C-13 isotopomers of CN -. These spectral signatures are identical to those reported earlier f or the one-electron-reduced cyanide adduct (cytochrome a(3) reduced), showing that the Fe-a3(2+)-CN- vibrational frequencies are independent of the redox states of the other three metal centers. Similarly, the Cu-B(2+) EPR signatures appear similar in both the one-electron- and t hree-electron-reduced cyanide adducts. On the other hand, the electron ic absorption spectra of ferrous a(3)-CN- show systematic red-shifts o f the alpha band as each of the other metal centers is reduced, and ot her, more subtle, differences in the electronic absorptions of the thr ee-electron-reduced and four-electron-reduced cyanide adducts are reve aled in the difference spectra. The relevance of these findings toward explaining the different cyanide binding and redox chemistry describe d herein and toward establishing the extent of structural analogy betw een the oxygen binding sites of the two proteins is discussed.