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Multiconformation continuum electrostatics analysis of the effects of a buried Asp introduced near heme a in Rhodobacter sphaeroides cytochrome c oxidase.

ABSTRACT: Cytochrome c oxidase (CcO) reduces O(2) to water via a series of proton-coupled electron transfers, generating a transmembrane electrochemical gradient. Coupling electron and proton transfer requires changing the pK(a) values of buried residues at each stage in the reaction cycle. Heme a is a key cofactor in the CcO electron transfer chain. Mutation of Ser44 to Asp has been reported [Mills, D. A., et al. (2008) Biochemistry 47, 11499-11509], changing the hydrogen bond acceptor from His102, the heme a axial ligand in Rhodobactor sphaeroides CcO. This adds an acidic residue to the CcO interior. The electrochemical behavior of heme a in wild-type and S44D CcO is compared using the continuum electrostatics program MCCE. The introduced, deeply buried Asp remains ionized at physiological pH only when the nearby heme is oxidized. Heme a reduction is now calculated to be strongly coupled to Asp proton binding, while with Ser44, it is weakly coupled to small protonation shifts at multiple sites, increasing the pH dependence in the mutant. At pH 7, the partially ionized Asp 44 is calculated to lower the heme redox potential by 50 mV as expected given the thermodynamics of coupled electron and proton transfers. This highlights an curious finding in the experimental results where a low Asp pK(a) is found together with a stabilized reduced heme. The stabilization of a heme oxidation in a model complex by a hydrogen bond to the axial His ligand calculated with continuum electrostatics and with density functional theory were in good agreement.

SUBMITTER: Zhang J

PROVIDER: S-EPMC3800205 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Multiconformation continuum electrostatics analysis of the effects of a buried Asp introduced near heme a in Rhodobacter sphaeroides cytochrome c oxidase.

Zhang Jun J Gunner M R MR

Biochemistry 20100901 37

Cytochrome c oxidase (CcO) reduces O(2) to water via a series of proton-coupled electron transfers, generating a transmembrane electrochemical gradient. Coupling electron and proton transfer requires changing the pK(a) values of buried residues at each stage in the reaction cycle. Heme a is a key cofactor in the CcO electron transfer chain. Mutation of Ser44 to Asp has been reported [Mills, D. A., et al. (2008) Biochemistry 47, 11499-11509], changing the hydrogen bond acceptor from His102, the h ...[more]

PMID: 20701325

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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.

Dataset Information

Multiconformation continuum electrostatics analysis of the effects of a buried Asp introduced near heme a in Rhodobacter sphaeroides cytochrome c oxidase.

Publications

Multiconformation continuum electrostatics analysis of the effects of a buried Asp introduced near heme a in Rhodobacter sphaeroides cytochrome c oxidase.

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