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Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.

ABSTRACT: Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1CF) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru(z)-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of <35 mM with the ruthenium photoexcitation technique in which k(12) became very rapid (3 x 10(9) M(-1) s(-1)) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ru(z)-23-c(552F) and c(1CF) was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 microM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ru(z)-39-Cc formed a tight 1:1 complex with cyt c(1CF) at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s(-1). A group of cyt c(1CF) variants in the presumed docking site were generated on the basis of information from the yeast cyt bc(1)-cyt c cocrystal structure. Kinetic analysis of cyt c(1CF) mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c(552F) and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.

SUBMITTER: Janzon J

PROVIDER: S-EPMC2645956 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.

Janzon Julia J Yuan Quan Q Malatesta Francesco F Hellwig Petra P Ludwig Bernd B Durham Bill B Millett Francis F

Biochemistry 20081201 49

Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1CF) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru(z)-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by ...[more]

PMID: 19006325

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Project description:Monospecific antibody to the respiratory NADH dehydrogenase from Paracoccus denitrificans was prepared by using as antigen specific immunoprecipitates containing NADH dehydrogenase which were excised from crossed-immunoelectrophoresis plates. The latter were run with selectively solubilized plasma membranes and antibodies against plasma membranes. The antibody immunoprecipitated NADH dehydrogenase from P. denitrificans membranes biosynthetically labelled with 14C and solubilized with a wide range of detergents. All immunoprecipitates contained the two subunits of Mr 48,000 and 25,000, in an approximate 1:1 stoichiometry, that had previously been assigned to NADH dehydrogenase. A polypeptide of Mr 46,000 in P. denitrificans membranes, previously shown to cross-react with a subunit-specific antibody to mitochondrial NADH dehydrogenase (complex I), was not detected in any immunoprecipitate. Under some conditions a third polypeptide, of Mr 31,000, was also detected, but in variable and non-stoichiometric amounts relative to the two other subunits. It was concluded that this polypeptide was incorporated into the immunoprecipitates as an artefact and that the polypeptides of Mr 48,000 and 25,000 are the sole polypeptides firmly identified in the NADH dehydrogenase. Flavoproteins were specifically radiolabelled by growth of P. denitrificans in the presence of [14C]riboflavin. Crossed immunoelectrophoresis of membranes from such cells showed that succinate dehydrogenase contained flavin, but that there was no detectable flavin in NADH dehydrogenase under these conditions. Analysis of excised immunoprecipitates of succinate dehydrogenase showed that flavin was covalently bound to a polypeptide of Mr 56,000. Flavin was retained by NADH dehydrogenase under mild conditions of detergent solubilization. Subsequent immunoprecipitation, followed by analysis of the acid-extracted flavin, established that FMN is a cofactor, in common with mitochondrial NADH-ubiquinone oxidoreductase (complex I).

Dataset Information

Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.

Publications

Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.

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