Biochemistry zyxwvut 1995, 34, zyxwvu 6335-6343 6335 Electron Transfer from Cytochrome zyxwv c to 8-Azido- ATP-Modified Cytochrome c Oxidaset3* Jian Lin, Shuguang Wu, and Sunney I. Chan* A. A. Noyes Laboratory zyxwvuts of Chemical Physics 127-72, California Institute of Technology, Pasadena, California 91125 Received November zyxwvutsr 18, 1994; Revised Manuscript Received February 21, 1995@ ABSTRACT: Bovine heart cytochrome c oxidase (CcO) has been modified by 8-azido-adenosine zy 5’- triphosphate (8-azido-ATP), and the electron-transfer activity from ferrocytochrome c to the modified CcO under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The kinetics of intermolecular electron transfer between the redox protein partners was shown to be reduced significantly. In addition, there is significant decrease in the binding affinity of the cytochrome zyxwvut c to the oxidase upon 8-azido-ATP modification. The 8-azido- ATP-modified CcO exhibited 50% of the intracomplex electron-transfer rate (ket) and 56% of the association constant (Ka) normally observed between cytochrome c and native CcO under otherwise identical conditions. Since the effective electron transfer rate constant is the product of ke, and Ka under nonsaturation conditions, the overall electron-transfer rate has been curtailed by over a factor of 2. Similar observations have been noted with the native CcO in the presence of 3 mM ATP. In contrast, the redox potential of neither CUA nor cytochrome a was altered upon 8-azido-ATP modification or in the presence of 3 mM ATP. Also, no gross structural changes at either the CUA or the cytochrome a site were noted, as evidenced by a lack of any spectral perturbations in the EPR signals from both of these centers. We conclude that ATP modulates the electron transfer from cytochrome c to CcO by interacting with the CcO and altering allosterically the docking. In this manner, ATP can ferrocytochrome c to cytochrome a and CUA. Cytochrome c oxidase (CcO),’ the terminal component of the mitochondrial respiratory chain, catalyzes the transfer of electrons from cytochrome c to dioxygen and couples electron transfer to the active transport of protons across the mitochondrial inner membrane. The steady-state oxidation of cytochrome c catalyzed by this enzyme shows distinctive biphasic behavior (Nicholls, 1964; Ferguson-Miller et al., 1976). The V,, values for the high- and low-affinity phases are 10-40 and 100-200 s-l, respectively (Errede & Kamen, 1978; Rosevear et al., 1980). Another interesting feature of the CcO-catalyzed reaction is that ATP, under physiologi- cal concentrations, influences the kinetics by abolishing, at least to a great extent, the high-affinity phase; in addition, V,, of the low-affinity phase is reduced (Ferguson-Miller et al., 1976). The latter effect is relevant to the control of turnover of the enzyme under physiological conditions since the rates fall within the physiological range. Over the years, there has been much evidence accumulat- ing to indicate that ATP regulates cellular respiration, especially the terminal electron-transport steps from cyto- ‘This work was supported by NIH Grant GM 22432 from the National Institute of General Medical Sciences, U.S. Public Health Service. * Contribution No. 9015. * To whom reprint requests should be addressed. @ Abstract published in Advance ACS Abstracts, May 1, 1995. ’ The subunit nomenclature of Kadenbach (Kadenbach et al., 1983) is used throughout this paper. Abbreviations: CcO, cytochrome c oxidase; ATP, adenosine 5’- triphosphate; 8-azido-ATP.8-azido-adenosine 5‘-triphosphate; 8-azido- ADP, 8-azido-adenosine 5’dphosphate; TNP-ATP, 2’ (or 3’)-0-(2,4,6- trinitropheny1)adenosine 5’-triphosphate; EDTA, ethylenediamine- tetraacetic acid; 5-DRF, 5-deazariboflavin;Hepes, 4-(2-hydroxyethyl)- 1 zyxwvutsrqp -piperazineethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane hydrochloride. 0006-2960/95/0434-6335$09.00/0 0 affect the branching of the electron input from chrome c to dioxygen mediated by CcO. Huther and Kadenbach (1986, 1987,1988) have proposed that the control is allosteric, with ATP binding to the oxidase modulating the details of the enzyme turnover, including the intramo- lecular electron-transferrates and the efficiency of biological energy transduction. Montecucco et al. (1986) have observed labeling of subunits IV and VI1 upon photo-cross-linking of CcO with 8-a~ido-[y-~~P]ATP. These workers have sug- gested that subunit IV and one of the subunit VI1 peptides provide the binding loci for the ATP. If so, this binding appears to exert a long-range conformational change in the structure of CcO, which affects the tertiary folding of subunit I1 and its efficacy to interact with cytochrome c (Bisson et al., 1987). Apparently, the negatively charged phosphate moiety of the ATP, more than the heterocyclic base linked to the sugar moiety, is the determinant here, since UTP has a kinetic effect on the oxidase activity similar to that of ATP (Bisson, et al., 1987). Several groups have attempted to mimic the specific effects of ATP on the steady-state kinetics of the CcO-catalyzed reaction by using covalently modified 8-azido-ATP CcO in the kinetics studies. The advantage of this approach is that the nonspecific effects of ionic strength on the docking of the redox protein partners can be obviated, particularly at high ATP concentrations. Free ATP also binds to cyto- chrome c under sufficiently high ATP concentrations, and this phenomenon may have deleterious effects on the interaction between cytochrome c and CcO (Craig & Wallace, 1991, 1993). Similar kinetic effects have been observed for 8-azido-ATP-modified CcO as for CcO in the 1995 American Chemical Society