Comparative Genomics and Site-Directed Mutagenesis Support the Existence of Only One Input Channel for Protons in the C-Family (cbb 3 Oxidase) of Heme-Copper Oxygen Reductases † James Hemp, ‡,§ Huazhi Han, ‡,| Jung Hyeob Roh, ⊥ Samuel Kaplan, ⊥ Todd J. Martinez, ‡,§ and Robert B. Gennis* ,‡,§,| Center for Biophysics and Computational Biology and Departments of Chemistry and Biochemistry, UniVersity of Illinois, 600 South Mathews Street, Urbana, Illinois 61801, and Department of Microbiology and Molecular Genetics, UniVersity of Texas Health Science Center, Houston, Texas 77030 ReceiVed April 7, 2007; ReVised Manuscript ReceiVed June 25, 2007 ABSTRACT: Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb 3 oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb 3 oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm. The heme-copper superfamily is structurally and catalyti- cally diverse, with members that perform either oxygen reductase or nitric oxide reductase reactions. The oxygen reductases are terminal oxidases in the respiratory chains of mitochondria and aerobic bacteria and archaea. These enzymes catalyze the reduction of O 2 to H 2 O utilizing a bimetallic active site that contains a high-spin heme and a copper ion. The oxygen reductases couple the chemical reaction to an electrogenic proton pump in which one proton is pumped across the membrane per electron transferred to the active site (1-3). The proton electrochemical gradient (protonmotive force) produced can be coupled to chemical synthesis, membrane translocation processes, and bacterial locomotion. The net reaction is where the subscripts IN and OUT refer to the cytoplasm and periplasm, respectively, for the bacterial and archaeal enzymes. † This work was supported by grants from the National Institutes of Health [HL16101 (R.B.G.) and GM15590-37 (S.K.)], from the Depart- ment of Energy [DE-FG02-87ER13716 (R.B.G.) and ER63232- 1018220-0007203 (S.K.)], and from the National Science Foundation [NSF-BES-04-03846 (T.J.M.)]. * Address correspondence to this author at the Department of Biochemistry, University of Illinois. E-mail: r-gennis@uiuc.edu. Fax: 217-244-3186. Tel: 217-333-9075. ‡ Center for Biophysics and Computational Biology, University of Illinois. § Department of Chemistry, University of Illinois. | Department of Biochemistry, University of Illinois. ⊥ University of Texas Health Science Center. O 2 + 8H + IN + 4e - / 2H 2 O + 4H + OUT 9963 Biochemistry 2007, 46, 9963-9972 10.1021/bi700659y CCC: $37.00 © 2007 American Chemical Society Published on Web 08/04/2007