Time-Resolved Step-Scan Fourier Transform Infrared Spectroscopy of the CO Adducts of Bovine Cytochrome c Oxidase and of Cytochrome bo₃ from Escherichia coli

Abstract

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.

Nanosecond electron tunneling between the hemes in cytochrome bo 3

“…The other phases reflect the multiphasic protein relaxation after CO photolysis in this enzyme (27), and the 3-s phase can be attributed to CO dissociation from the Cu B center (18,23), which lies next to the oxygen-binding heme and is the first site of CO binding after photolysis (28,29). Therefore, only the nanosecond phase can be ascribed to true electron tunneling.…”

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

“…2, trace E). The spectra of the CO-bound forms of the enzyme exhibit a major peak at 13 CO, pH 7.5 (C). The CO-bound forms of cytochrome aa 3 from P. denitrificans (D) and the mammalian counterpart (E) at pD ϭ 7.5 are included for comparison.…”

“…The Fourier transform infrared (FTIR) spectra of the CObound heme-copper oxidases have revealed several characteristics of the binuclear pocket, including, from the frequencies of the C-O stretching modes of heme iron and Cu B , the identity of the metal to which the CO is bound, as well as the interactions between the axial ligands and the heme and/or the Cu B environment (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Cryogenic techniques have also been used successfully in conjunction with FTIR to cryotrap metastable intermediates by first freezing the CO-bound protein at cryogenic temperatures and then photodissociating the CO (6 -10).…”

“…Cryogenic techniques have also been used successfully in conjunction with FTIR to cryotrap metastable intermediates by first freezing the CO-bound protein at cryogenic temperatures and then photodissociating the CO (6 -10). Recently, the comparison between the cryotrapped and room temperature intermediates of photodissociated aa 3 from mammalian and bo 3 from Escherichia coli was reported, and the results were surprising (13). Although the frequencies of the major heme-CO and Cu B 1ϩ -CO bands are very similar between the cryogenic and the room temperature spectrum, features due to a change in absorbance of the CϭO stretch of the carboxylic acid side chain of aspartic or glutamic acids are very different.…”

Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS

“…Such measurements, e.g. by tr-FTIR were previously performed after photodissociation of CO adducts of the CcO [24][25][26][27][28][29][30][31][32][33]. Photodissociation of CO adducts was combined with oxidation of the enzyme by O 2 or caged O 2 [34,35], yielding tr-FTIR measurements in the time scale of seconds [36,37].…”