Possible proton relay pathways in cytochrome c oxidase.

Abstract

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Aspl32Asn/Ala), an exterior loop between helices IX and X (His4l]Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity. Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Aspl32 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-I-I loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.Cytochrome c oxidase (EC 1.9.3.1), a key enzyme in aerobic energy metabolism, reduces oxygen to water, yielding substantial energy that drives the formation of a proton gradient; however, the mechanism of coupling between oxygen reduction and proton translocation remains obscure.Recognition of the strong homology between mitochondrial and bacterial enzymes (1, 2) has stimulated the application of molecular genetic tools to the analysis of the oxidase mechanism. The genes for cytochrome c oxidase from Rhodobacter sphaeroides have been cloned, sequenced, deleted, and reintroduced into the bacterium, and sequence comparisons reveal a high degree of homology with the three mitochondrially encoded subunits of mammalian oxidase (3-7). Extensive sitedirected mutagenesis of the largest subunit, COX I, has permitted the assignment of the ligands for the three redox active metal centers, heme a, heme a3, and CUB (7-10), suggesting that all three metal centers are located in COX I toward the outer side of the membrane, while substrate and pumped protons come from the inside (11). Thus some kind of proton channel or relay system is required to convey protons to the site of oxygen reduction, the heme a3-CuB center, and beyond. It is reasonable to look for residues involved in proton pumpingThe publication costs of this article were defrayed in part by page charge payment. This article m...

Fatty acids stimulate activity and restore respiratory control in a proton channel mutant of cytochrome c oxidase

“…D132 is a substantial distance from the oxygen-reactive site and mutation of the residue does not perturb the spectral properties of the metal centers [4]. Similar effects are seen when the corresponding residue in Escherichia coli cytochrome bo3 is mutated [5,6]).…”

“…Similar effects are seen when the corresponding residue in Escherichia coli cytochrome bo3 is mutated [5,6]). Another effect of the D132N/A mutations is that the reconstituted enzymes show anomalous responses to uncouplers and ionophores [4]. Valinomycin and CCCP, both singly and in combination, inhibit instead of stimulating turnover in reconstituted enzyme.…”

Reduction of CO2 to Methanol Catalyzed by a Biomimetic Organo-Hydride Produced from Pyridine

“…This view is also supported by the prior configuration of the water molecules in the aqueous solution solvating the reactant complex and the widespread occurrence of proton relays in other processes, [55][56][57]59,60,[64][65][66][67] including water oxidation 54,58 and enzymatic reactions. [61][62][63] In any event, the ∆G ‡ HT's reported in Table 1 show that the reaction is viable even without involvement of any proton relay chain.…”

“…In addition to stabilizing the TS, these water molecules also intimately participate in the reaction by acting as a proton relay chain during the proton transfer event. 47,[54][55][56][57][58][59][60][61][62][63][64][65][66][67] We calculate vibrational force constants at the M06/6-31+G** level of theory to: 1) verify that the reactant and product structures have only positive vibrational modes, 2) confirm that each TS has only one imaginary mode and that it connects the desired reactant and product structures via Intrinsic Reaction Coordinate (IRC) calculations, and 3) compute entropies, zero-point energies (ZPE) and thermal corrections included in the reported free energies at 298K.…”

“…99 In particular, using a hybrid explicit/implicit solvent model, we calculated low enthalpic barriers with respect to the complexed reactants of 13.6-18.5 kcal/mol (depending on the number of solvating waters) for PyCOOH 0 formation via a proton relay mechanism; the importance of proton relays have been extensively described in assorted chemical reactions. [54][55][56][57][58][59][60][61][62][63][64][65] Charge analysis on CO2 and PyH 0 along the reaction coordinate reveals that PyH 0 's propensity to recover its aromaticity drives the sequence of ET to CO2 followed by PT (mediated by a proton relay) to ultimately form PyCOOH 0 . 47,100 While this particular reaction is not of direct interest in the present work, 101 we will see that the themes of aromaticity recovery and proton relay mechanisms also prove to be important for our three HTPT step reduction of CO2 to CH3OH.…”