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Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase.

ABSTRACT: Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ?5,000. Here, we show that proton uptake at full speed (?10(4) s(-1)) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn(2+) addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn(2+) binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

SUBMITTER: Johansson AL

PROVIDER: S-EPMC3670322 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase.

Johansson Ann-Louise AL Högbom Martin M Carlsson Jens J Gennis Robert B RB Brzezinski Peter P

Proceedings of the National Academy of Sciences of the United States of America 20130514 22

Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidas ...[more]

PMID: 23674679

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Project description:Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O(2) to H(2)O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides . Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200Ile, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P(R) ? F and F ? O transitions (P(R) is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O(2), F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the P(R) ? F transition is shown to be essentially pH independent up to pH 12 (i.e., the apparent pK(a) of Glu286 is increased from 9.4 by at least 3 pK(a) units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO.

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Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase.

Publications

Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase.

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