Spectroscopic characterization of cytochrome ba3, a terminal oxidase from Thermus thermophilus: Comparison of the a3/CuB site to that of bovine cytochrome aa3

Abstract

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.

Fourier Transform Infrared (FTIR) and Step-scan Time-resolved FTIR Spectroscopies Reveal a Unique Active Site in Cytochromecaa3 Oxidase from Thermus thermophilus

“…Also, the strength of the proximal histidine H-bonding interaction affects the strength of both the Fe-C and C-O bonds that are further influenced by the Cu B distal environment (15,17). The Fe-His vibration of heme a 3 in caa 3 has a frequency at 211 cm Ϫ1 (4), which is close to those found in other aa 3 type oxidases and to the high frequency Fe-His (193 and 208 cm Ϫ1 ) conformer of ba 3 (18). Taken together, from the present FTIR and previous resonance Raman (RR) data (4) it appears that the distal to the heme a 3 environment has more direct control on the vibrational properties of the heme a 3 2ϩ -CO complex.…”

Resonance Raman, Infrared, and EPR Investigation on the Binuclear Site Structure of the Heme-Copper Ubiquinol Oxidases from Acetobacter aceti: Effect of the Heme Peripheral Formyl Group Substitution

“…CO-reduced 1644 CN-reduced 1671 oxidized 1671 CN-oxidized (Rousseau et al, 1988) 1666 NO-reduced (Heibel et al, 1993b) 1664.7 reduced 1673.6 oxidized Sulfolobus acidocaldarius (Heibel et al, 1993a) 1668 reduced 1672 reduced 5cHS 1673 oxidized 6cHS 1666 oxidized 5cHS cytochrome ba 3 Thermus thermophilus (Oertling et al, 1994) 1669 reduced 1657…”

Redox-linked transient deprotonation at the binuclear site in the aa 3 -type quinol oxidase from Acidianus ambivalens : Implications for proton translocation

“…However, a time-dependent change subsequently occurs in the Raman spectrum, involving a slow shift in frequency of the formyl stretching mode of heme a 3 , from 1,660 to 1,667 cm Ϫ1 . Immediately after reduction, the frequency of the a 3 formyl stretching mode (1,660 cm Ϫ1 ) observed in this enzyme is the lowest among the heme a 3 -containing oxidases, where this mode occurs in the 1,662 to 1,669 cm Ϫ1 range (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The absence of spectral changes in the other modes, including the oxidation state marker, 4 , at 1,355 cm Ϫ1 (data not shown), indicates that the oxidation, coordination, and spin states are not changing as a function of time after reduction in either of the hemes.…”