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

Abstract

Acetobacter aceti produces two different terminal ubiquinol oxidases (cytochromes a1 and o) depending on the culture conditions. Two types of oxidases share a common protein moiety but with different heme components at the binuclear center (heme A for cytochrome a1 and heme O for cytochrome o). We investigated the structure of the binuclear site of the two oxidases using resonance Raman, Fourier transform-infrared (FT-IR), and EPR spectroscopies to clarify the interactions of heme A formyl group with protein moiety. We found that the overall architecture and the electronic configuration at the binuclear center in the oxidized state seem to be well conserved irrespective of the heme peripheral group at position 8, except for the azide-inhibited state. In contrast, we observed great variations in the C-N stretching frequency and cyanide-binding affinity in the CN-reduced state, in addition to multiple C-O stretching bands in the CO-reduced state. Present and previous studies suggest that the conformational flexibility of the binuclear center in the reduced ligand-bound state may be a common feature among the heme-copper oxidase superfamily. In the CN-reduced state, a hydrogen bond network may be formed among the formyl group, water molecule(s), and the surrounding amino acid residue(s). This network may be very important to maintain proper orientations of the distal amino acid residues and/or the CuB1+ ion relative to the cyanide ion bound to the ferrous heme iron and could play a critical role for the high affinity in cyanide binding.

Fourier‐transform infrared studies on azide‐binding to the binuclear center of the Escherichia coli bo‐type ubiquinol oxidase1

“…The clear difference in the peak positions of the major bound azide band between bovine cytochrome c oxidase (2051 cm −1 ) and the ubiquinol oxidases (2041 cm −1 ) suggests such distinctions. Indeed, a similar difference was previously observed for the bridging cyanide stretching vibrations (2152 versus 2146 cm −1 , respectively) [7,11,13] and was explained due to the difference in the Cu B ‐N‐C bond angle and Cu B ‐N bond distance [14].…”

“…Substitutions with asymmetrically 15 N‐labelled azides ( 15 N 14 N 14 N and 15 N 15 N 14 N), however, did not cause a splitting of the infrared band. Further, we found that a major bound azide band at 2041 cm −1 of the Acetobacter aceti ubiquinol oxidase did not cause a splitting upon the 15 N isotope substitutions for both ba ‐ and bo ‐type enzymes in the air‐oxidized state [11]. These properties are very similar to the corresponding band at 2051 cm −1 of the azide‐inhibited air‐oxidized bovine cytochrome c oxidase [4,8].…”

“…In the azide‐inhibited air‐oxidized form of bovine cytochrome c oxidase, EPR signals from the heme‐copper binuclear site were not so much different from those of the air‐oxidized form [4]. On the other hand, for the azide‐inhibited E. coli [7] and A. aceti [11] ubiquinol oxidases, an EPR signal ( g *=3.2) derived from an integer spin system produced by a spin‐spin exchange coupling at the binuclear site appeared. Although the comparison of the coupling efficiency via a bridging azide is difficult to estimate, it is clear that the geometry of the azide‐bridging might be different from each other.…”

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.…”

Insight into protein structure and protein-ligand recognition by Fourier transform infrared spectroscopy

“…The interesting recognition phenomena are the binding of dioxygen in the binuclear reaction center and the transfer of electrons and protons to O 2 (Yamazaki et al, 1999). The binuclear reaction center has been extensively characterized also by FTIR using CO, NO, CN À and N 3 À as spectroscopic probes (Tsubaki et al, 1996(Tsubaki et al, , 1997Zhao et al, 1994). Depending on the redox state of the different redox centers within the bovine heart cytochrome c oxidase, the stretch frequency of the CO ligand is observed between 1959 and 1965 cm À1 (Yoshikawa et al, 1977;Yoshikawa and Caughey, 1982).…”