Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel.
Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined. Previous mutagenesis studies of the cytochrome bo(3) quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel. The current work examines the properties of mutants of this conserved glutamate in the oxidase from R. sphaeroides (E101(II)I,A,C,Q,D,N,H) and residues in the immediate vicinity of E101(II). It is shown that virtually any substitution for E101(II), including E101(II)D, strongly reduces oxidase turnover (to 8-29%). Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a(3) prior to the reaction with O(2). These are phenotypes expected of K-channel mutants. It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101(II) in subunit II.
Tomson
Farol L
FL
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Morgan
Joel E
JE
Gu
Guoping
G
Barquera
Blanca
B
Vygodina
T V
TV
Gennis
Robert B
RB
D019943:Amino Acid Substitution / Q000235:genetics; D017124:Conserved Sequence; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D054328:Proton Pump Inhibitors; D017494:Proton Pumps / Q000737:chemistry*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D016877:Oxidants; D021122:Protein Subunits; D054328:Proton Pump Inhibitors; D017494:Proton Pumps; D011522:Protons; C027728:heme a; D018698:Glutamic Acid; D006418:Heme; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV