Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase I. BIOCHEMICAL, SPECTRAL, AND KINETIC CHARACTERIZATION OF SURFACE MUTANTS IN SUBUNIT II OF RHODOBACTER SPHAEROIDESCYTOCHROME aa 3

Abstract To determine the interaction site for cytochromec (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp143, was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc2, showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K mvalues, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp143 severely inhibit activity without changing theK m for Cc or disturbing the adjacent CuA center. From these data, we identify a Cc binding area on CcO with Trp143 and Asp214 close to the site of electron transfer and Glu148, Glu157, and Asp195 providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem.274, 38042–38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051–38060).