To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na ؉ /K ؉ -ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na ؉ /K ؉ -ATPase ␣-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na ؉ /K ؉ -ATPase or low affinity ATP inhibition of K ؉ -para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K 0.5 . However, both the affinities for the K480E were reduced to ϳ1/20. The turnover number of the Na ؉ /K ؉ -ATPase and the apparent affinity for Na ؉ and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K ؉ -pNPPase and the apparent affinity for K ؉ were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.

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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 ...