Cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of ATP. The catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and O2 reduction. These events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and protonation states. We have used IR difference spectroscopy to investigate such modulations for the functional intermediate states E, R2,Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the Pm states. The consecutive deprotonation and reprotonation of E286 twice within one catalytic turnover illustrates the role of this residue as a proton shuttle. In addition, the spectra point toward deprotonation of a redox-active tyrosine, plausibly Y288, in the F intermediate. Structural insights into the molecular mechanism of catalysis based on the subtle molecular changes observed with IR difference spectroscopy are discussed.

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.

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flowexchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions. ß

Infrared Spectroscopy is a powerful tool in investigating protein dynamics on an atomic level. Time-resolved Fourier-transform infrared spectroscopy (FTIR) was particularly helpful in elucidating individual proton transfer steps in bacteriorhodopsin (1). In an attempt to study the electron-driven proton translocation of oxidases we applied FTIR spectroscopy to cytochrome c oxidase from bovine heart and from R. spbaeroides. Preliminary experiments on fully-reduced and CO saturated enzymes exhibited very small but distinct spectral changes in the mid-infrared region after flashing-off the C O by a nanosecond laser pulse. Vibrational changes were recorded with the rapid-scan and the stepscan technique (present time-resolution: 100 cs).At 253K, CO photodissociation leads to a large negative band at 1963 cml. Transfer of C O from heme a3 to CUB resulted in a positive band at 2062 cm-l (for bovine heart oxidase). Accompanying those changes, highly resolved spectra in the region below 1800 cm-l indicated specific changes in the vinyl and formyl vibrations of the heme as well as vibrations of the binuclear center (2). Moreover, changes in frequencies of amino acid side chains could also be detected and resolved in time. Spectra were measured in H 2 0 and D 2 0 for the mammalian and compared to the bacterial enzyme.The Na,K-ATPase is an integral membrane enzyme which establishes and maintains the Na + and K+ electrochemical gradient across the plasma membrane. The Chemical modification of Lys-501 in the pig kidney Na,K-ATPase with FITC(fluorescein-5'-isothiocyanate) inhibits transphosphorylation of terminal phosphate of ATP to Asp-369 and reduced the apparent affinity for ATP. To investigate the role of this residue in Na,K-ATPase activity, Lys-508 in rat kidney Na,K-ATPase (correspond to Lys-501 in pig kidney Na,K-ATPase) was changed to Ala and Glu by site-specific mutagenesis, and the resultant enzymes were stably expressed in HeLa cells.Eikenella corrodens, a Gram-negative rod, was originally isolated fiom the human oral cavity and is an opportunist pathogen. It is a fastidious bacterium with sophisticated requirements for its culture in axenic medium; its growth in complex liquid medium is poor and not easily reproducible.Here we report our preliminary results concerning to some bioenergetic properties of E. cotrodens ATCC 23834 obtained fiom microaerophilic cultures (static) in the modified BMI liquid complex medium (Allaker et d., FEMS Microbiol. Lett. 123: 69-74, 1994). The aim of the work is to get insight into the composition and organization of the respiratory system of E. corrodens cultured under limited oxygen.We found that purifies cell membranes were able to oxidize NADH, succinate, ascorbate plus TMPD and formate. The spectroscopic analysis of membrane particles at room temperature and 77 K revealed the presence of c and &type cytochromes that were reducible by dithionite and physiological electron donors. CO-difference spectra suggested the presence of a cytochrome u-type oxidase however this could net be ...