Transient Binding of CO to CuB in Cytochrome c Oxidase Is Dynamically Linked to Structural Changes around a Carboxyl Group: A Time-Resolved Step-Scan Fourier Transform Infrared Investigation

Abstract

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.

Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications

“…CO also binds to proteins that contain other transition metals at the active site, e.g., copper, and by doing so interferes with their functions. Examples include heme-copper cytochrome c oxidase (Heitbrink et al, 2002), the half-apo derivative of peptidylglycine monooxygenase (Jaron and Blackburn, 2001), and cytochrome bo quinol oxidase (Ciccognani et al, 1992). Although CO's interaction with heme proteins underlies most of its physiological effects, it also targets the signaling pathways that are not categorized as heme proteins, such as different ion channels.…”

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

“…The pK a of E286 is high [9.4 (16); see also refs. [17][18][19] and presumably finely tuned to deliver protons to the different locations in the correct sequence. In addition, the protonpumping machinery requires cycling between states with alternating access for protons either to the negative or to the positive side of the membrane (''gating'').…”

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

“…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).…”