Article

Communication between L-galactono-1,4-lactone dehydrogenase and cytochrome c.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and University of Sevilla, cicCartuja, Américo Vespucio 49, 41092, Seville, Spain.
FEBS Journal (Impact Factor: 3.99). 02/2013; DOI: 10.1111/febs.12207
Source: PubMed

ABSTRACT L-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed formation and stabilization of the GALDH semiquinone anionic species (GALDH(SQ) ). GALDH(SQ) oxidation by Cc exhibited a non-linear dependence on Cc concentration consistent with a kinetic mechanism involving protein-partner association to form a transient bimolecular complex prior to the electron transfer step. Oxidation of GALDH(SQ) by Cc was significantly impaired at high ionic strength, revealing the existence of attractive charge-charge interactions between both reactants. Isothermal titration calorimetry showed that GALDH weakly interacts with both oxidized and reduced Cc. Chemical shift perturbations for (1) H and (15) N nuclei of Cc, arising from the interactions with unlabelled GALDH, were used to map the interacting surface of Cc. For Arabidopsis Cc and yeast Cc, similar residues are involved in the interaction with GALDH. These residues are confined to a single surface surrounding the heme edge. The range of chemical shift perturbations for the physiological Arabidopsis Cc-GALDH complex is larger than that of the non-physiological yeast Cc-GALDH complex, indicating that the former complex is more specific. In summary, the results point to a relatively low-affinity GALDH/Cc interaction, similar for all partner redox states, involving protein-protein dynamic motions. Evidence is also provided that Cc utilizes a conserved surface surrounding the heme edge for the interaction with GALDH and other redox partners. © 2013 The Authors Journal compilation © 2013 FEBS.

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