IDENTITY OF THE AXIAL LIGAND OF THE HIGH-SPIN HEME IN CYTOCHROME-OXIDASE - SPECTROSCOPIC CHARACTERIZATION OF MUTANTS IN THE BO-TYPE OXIDASEOF ESCHERICHIA-COLI AND THE AA3-TYPE OXIDASE OF RHODOBACTER-SPHAEROIDES

Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases. The se enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms. Two such hem e-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli. Th ese enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center. Energy conservation is accomplished by coupling ele ctron transfer through the metals of the oxidases to proton translocat ion across the cellular membrane. The Rb. sphaeroides and E. coli enzy mes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a) , as well as additional histidine residues which are probable ligands for copper (CUB). However, the histidine that binds the heme of the bi nuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419). Additional characterization by Four ier transform infrared spectroscopy of the CO-bound forms of the E. co li enzyme in which His284 is replaced by glycine or leucine demonstrat es that these mutations cause only subtle changes to CO bound to the h eme of the binuclear center. Resonance Raman spectroscopy of the Rb. s phaeroides enzyme in which His284 is replaced by alanine shows that th e iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419. These results demons trate that His284 is not the heme a3 ligand. Therefore, the remaining conserved histidine within subunit I of the oxidases (His419) is propo sed to be the heme a3 ligand. In this model, the axial ligands of the two hemes are located within a single helix and thus are connected by a pathway of covalent bonds. The implications of this model on the con trol of electron transfer through the enzyme are discussed.