Dataset Information

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

ABSTRACT: The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.

SUBMITTER: Marantz Y

PROVIDER: S-EPMC21120 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

Marantz Y Y Nachliel E E Aagaard A A Brzezinski P P Gutman M M

Proceedings of the National Academy of Sciences of the United States of America 19980701 15

The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the b ...[more]

PMID: 9671722

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Project description:The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an approximately 15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the F --> O transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O(2) shows approximately 200-fold inhibition of the F --> O step (k approximately 2 s(-1) at pH 8) which is not compatible with enzyme turnover ( approximately 30 s(-1)). Presumably, an abnormal intermediate F(deprotonated) is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the F --> O transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H(2)O(2), decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Deltapsi generation coupled to the flash-induced F --> O transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a(3) from the Fe(4+) horizontal lineO(2-) state to the Fe(3+)-OH(-) state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the F --> O step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of approximately 10 and approximately 40 mus and a magnitude ratio of approximately 3:2. The 10 mus phase matches vectorial electron transfer from Cu(A) to heme a, whereas the 40 mus component is assigned to intraprotein proton displacement across approximately 20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D-channel mutants as well as with cyanide-inhibited wild-type oxidase. The finding helps to reconcile the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme ( approximately 30%) with the net electrogenicity of the F --> O transition coupled to transmembrane transfer of two charges per electron.

Project description:The aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides utilizes two proton-input channels to provide all the protons for chemistry (water formation) and proton pumping. The D-channel is responsible for the uptake of all pumped protons, four protons per O(2). Several substitutions of either N139 or N207, near the entrance of the D-channel, were previously reported to decouple the proton pump from oxidase activity. In this work, the characteristics of additional mutations in this region of the protein (N139, N207, N121, and S142) are determined to elucidate the mechanism of decoupling. With the exception of the substitution of a large, hydrophobic residue (N139L), all the mutations of N139 resulted in an enzyme with high oxidase activity but with a severely diminished proton pumping stoichiometry. Whereas N207D was previously shown to be decoupled, N207A and N207T exhibit nearly wild-type behavior. The new data display a pattern. Small, nonionizable substitutions of N139 or N121 result in decoupling of the proton pump but maintain high turnover rates. These residues are directly hydrogen bonded to two water molecules (Water6574 and Water6584) that are part of the single-file chain of water molecules within the D-channel leading to E286 at the top of the channel. The data suggest that the integrity of this water chain within the D-channel is critical for rapid proton transfer. The mechanism of decoupling is most likely due to the slowing of the rate of proton delivery below a threshold that is required for protonation of the putative proton loading site. Protons delivered outside this time window are delivered to the active site where they are consumed in the formation of water. The rate of proton delivery required to protonate the pump site must be significantly faster than the rate of delivery of protons to the catalytic site. For this reason, mutations can result in decoupling of the proton pump without slowing the catalytic turnover by the enzyme.

Dataset Information

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

Publications

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

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