The proton pumping pathway of bovine heart cytochrome c oxidase

Abstract

X-ray structures of bovine heart cytochrome c oxidase have suggested that the enzyme, which reduces O(2) in a process coupled with a proton pumping process, contains a proton pumping pathway (H-pathway) composed of a hydrogen bond network and a water channel located in tandem across the enzyme. The hydrogen bond network includes the peptide bond between Tyr-440 and Ser-441, which could facilitate unidirectional proton transfer. Replacement of a possible proton-ejecting aspartate (Asp-51) at one end of the H-pathway with asparagine, using a stable bovine gene expression system, abolishes the proton pumping activity without influencing the O(2) reduction function. Blockage of either the water channel by a double mutation (Val386Leu and Met390Trp) or proton transfer through the peptide by a Ser441Pro mutation was found to abolish the proton pumping activity without impairment of the O(2) reduction activity. These results significantly strengthen the proposal that H-pathway is involved in proton pumping.

Fig. 1.

Schematic representation of the H-pathway of oxidized bovine heart CcO. Hydrogen bond network extends from Arg-38 to Asp-51 including a peptide bond between Tyr-440 and Ser-441. The dotted line represents the hydrogen bond. The water channel (represented by the gray area) allows access of water molecules in the negative side space to the formyl group of heme a, which is hydrogen bonded to Arg-38. Heme a also interacts with the H-pathway via the other hydrogen bond between the propionate group and water (represented by the black sphere). Mutation sites are highlighted in red.

Fig. 2.

Proton transfer through the peptide bond between Tyr-440 and Ser-441. (a) Mechanism of proton transfer through the peptide bond. Protonation of the oxygen of the peptide bond by proton from the negative side yields the imidic acid intermediate (C(OH)N⁺H) (8), followed by removal of the imidic acid proton by D51 to form the enol tautomer of the peptide (C(OH)N) (6). The enol form is then tautomerized back to the keto form (CO-NH) because the latter is more stable. (b) Protonation of the peptide bond by proton from the negative side in the Ser441Pro mutant. (c) Prediction of the conformation of the Ser441Pro mutant. The purple structures show the conformational changes of the oxidized form induced by the Ser441Pro mutation as predicted by X-PLOR analysis. The dark blue structure denotes the conformational change occurring upon reduction of the enzyme, wherein no influence by the mutation is detectable. The red dotted lines indicate hydrogen bonds, and the red, yellow, and light blue structures denote oxygen, carbon, and nitrogen atoms, respectively.

Fig. 3.

Immunoblot analyses of CcO of HeLa cells. (a) Immunoblot analyses of CcO fractionated by blue-native PAGE of solubilized mitochondrial fractions. Solubilized mitochondria (70 μg) from HeLa cells harboring the expression vector without the bovine subunit I gene (lane 1) and with that of wild type (lane 2), Ser441Pro (lane 3), and Val386Leu/Met390Trp (lane 4) were fractionated on blue native PAGE (10). CcO complex electrophoresed at 210 kDa was demonstrated by immunoblot analysis to be positive for the bovine subunit I specific antibody (6), indicating that the bovine subunit I formed the hybrid enzyme with the other human subunits. (b) Immunoblot analysis of human subunit I of CcO. Dodecylmaltoside (1.4%)-solubilized CcO (2.5 pmol) from mitochondrial preparations was fractionated by SDS/PAGE. Mitochondrial samples were obtained from HeLa cells harboring the expression vector without the bovine subunit I gene (lanes 1, 3, and 5), and with that of wild type (lane 2), Ser441Pro (lane 4) and Val386Leu/Met390Trp (lane 6). Human subunit I was detected by immunoblot analysis using the specific antibody (6). When bovine subunit I was expressed simultaneously, the intensity of the subunit I band detected by human subunit I antibody was ≈20% that of the pure human subunit I, indicating that ≈80% of CcO contained bovine subunit I.

Fig. 4.

Absorption spectra of mitochondrial preparations. Visible difference absorption spectra of samples before and after treatment with a slight excess of dithionite. The mitochondrial preparations were isolated from HeLa cells harboring the expression vectors without the bovine heart cytochrome c subunit I gene (cnt.) and with that of the wild type (wt), Ser441Pro (441P), and Val386Leu/Met390Trp double mutant (LW). The mitochondrial preparations were treated with 1.4% dodecylmaltoside before obtaining absorption measurements. The protein concentration for each measurement was ≈1 mg/ml in 100 mM HEPES-KOH buffer, pH 7.0, containing 0.14% dodecylmaltoside in a 1-cm cuvette.

Fig. 5.

Ferrocytochrome c oxidation and proton pumping of bovine/human hybrid CcO. The experimental conditions are described in Materials and Methods. (a–c) Ferrocytochrome c oxidation monitored at 550 nm, the absorption peak characteristics of cytochrome c in the ferrous state. (d–f) Proton ejection from mitoplasts by CcO after addition of ferrocytochrome c. The hybrid enzymes investigated were wild type (a and d), Ser441Pro mutant (b and e), and Val386Leu/Met390Trp double mutant (c and f). Proton ejection by the wild-type hybrid enzyme (d) in the initial 15 s after addition of ferrocytochrome c is followed by a decrease in proton concentration due to the proton back-leak into the matrix space. Weak acidification in the initial 10 s for the Ser441Pro mutant (e) and the double mutant (f) is induced by 20% residual human CcO. +FCCP denotes the proton ejection trace obtained in the presence of FCCP, a proton ionophore. The results of the two mutant enzymes in (b, c, e, and f) were obtained at the enzyme concentration 20% higher than those of the wild-type enzyme.

Fig. 6.

Possible proton acceptor sites near Asp-51 in bovine heart CcO. (Upper) Space filling model of bovine heart CcO at 1.9 Å resolution in the reduced state. Red and blue spheres represent oxygen and nitrogen atoms of acidic and basic amino acid side chains. Asp-51 and three possible proton acceptors (H138, D50, and D445) from Asp-51 are indicated by arrows. The distances from the Asp-51 carboxyl group are indicated by the digits in Å unit on the double-headed arrows. (Lower) Hydrogen bond network connecting Asp-51 with the possible proton accepting sites (H138, D50, and D445). Dotted lines denote hydrogen bonds. The double headed dotted arrow shows a possible pathway where either one of the two water molecules can migrate to the other water molecule within the hydrogen-bond distance for proton transfer.