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A Copper Protein and a Cytochrome Bind at the Same Site on Bacterial Cytochrome C Peroxidase† Sofia R

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A Copper Protein and a Cytochrome Bind at the Same Site on Bacterial Cytochrome C Peroxidase† Sofia R 14566 Biochemistry 2004, 43, 14566-14576 A Copper Protein and a Cytochrome Bind at the Same Site on Bacterial Cytochrome c Peroxidase† Sofia R. Pauleta,‡,§ Alan Cooper,⊥ Margaret Nutley,⊥ Neil Errington,| Stephen Harding,| Francoise Guerlesquin,3 Celia F. Goodhew,‡ Isabel Moura,§ Jose J. G. Moura,§ and Graham W. Pettigrew‡ Veterinary Biomedical Sciences, Royal (Dick) School of Veterinary Studies, UniVersity of Edinburgh, Summerhall, Edinburgh EH9 1QH, U.K., Department of Chemistry, UniVersity of Glasgow, Glasgow G12 8QQ, U.K., Centre for Macromolecular Hydrodynamics, UniVersity of Nottingham, Sutton Bonington, Nottingham LE12 5 RD, U.K., Unite de Bioenergetique et Ingenierie des Proteines, IBSM-CNRS, 31 chemin Joseph Aiguier, 13402 Marseilles cedex 20, France, Requimte, Departamento de Quimica, CQFB, UniVersidade NoVa de Lisboa, 2829-516 Monte de Caparica, Portugal ReceiVed July 5, 2004; ReVised Manuscript ReceiVed September 9, 2004 ABSTRACT: Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a Kd of 16.4 µMat25°C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and 1H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 Å. This is consistent with the results of 1H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme. Electron-transfer processes have to be both fast and in structure and mechanism and afford a parallel model specific. Specificity requires buried redox centers and tight system for investigation (3-5). The active form of bacterial binding with accurate recognition of surfaces, but speed cytochrome c peroxidase is a mixed valence state in which requires exposed redox centers and binding that is loose an electron-transferring heme contains Fe(II) and a peroxi- enough to allow prompt dissociation of the product complex. datic heme contains Fe(III) (6, 7). The enzyme supplies two Reconciliation of the requirements for specificity and speed electrons for the reduction of the substrate, hydrogen can be achieved in a model involving fluid and transient peroxide, to water, and thus two electrons are required to encounter complexes. Features of this model include elec- restore the active form of the enzyme after turnover. We trostatic preorientation of redox donors and lateral search have shown that the cytochrome c peroxidase from Para- within transient complexes for favorable electron conduction coccus pantotrophus can bind cytochrome c550 (the physi- routes to largely or completely buried redox centers (1). The ological electron donor) at a site close to the electron- transient nature of such complexes means that cocrystallog- transferring heme but that horse cytochrome c (a non- raphy is often unsuccessful and other means have to be physiological electron donor) binds preferentially at a dif- employed to characterize them. ferent site (8, 9). In addition, we have found that the The eukaryotic cytochrome c peroxidase from yeast has peroxidase can accommodate both horse cytochrome c and become a model system for the study of biological electron cytochrome c550 in a ternary complex (10). Although this transfer (2). Bacterial cytochrome c peroxidases are distinct is a nonphysiological complex, it does raise the possibility that the enzyme is designed to accommodate two redox † This work was supported with a PhD grant from Fundac¸a˜o para a donors at the same time. Cieˆncia e Tecnologia (No. BD/18297/98) to S.R.P. and a BBSRC grant Pseudoazurin is a small copper protein that has been shown to G.W.P. to substitute for cytochrome c550 in the reduction of the * Corresponding author. Tel: 44-131-650-6135. Fax: 44-131-165- 6576. E-mail: [email protected]. enzyme nitrite reductase (cytochrome cd1)inParacoccus ‡ University of Edinburgh. pantotrophus LMD 82.5(11). We have shown that the § Universidade Nova de Lisboa. pseudoazurin gene is present and is identical in the closely ⊥ University of Glasgow. | University of Nottingham. related organism Paracoccus pantotrophus LMD 52.44, 3 IBSM-CNRS. which we have used for our studies on cytochrome c 10.1021/bi0485833 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/29/2004 Binding of Pseudoazurin to Bacterial Cytochrome c Peroxidase Biochemistry, Vol. 43, No. 46, 2004 14567 peroxidase (1). Cytochrome c peroxidase has comparable correspond to a pseudoazurin binding to each monomer of activities when cytochrome c550 or pseudoazurin are used the dimeric protein. as electron donors (1). Both these proteins have pronounced Microcalorimetry. Protein solutions were equilibrated with charge asymmetry with a ring of lysines surrounding a the appropriate buffer by molecular exclusion chromatog- relatively hydrophobic front face at the center of which is raphy on Sephadex G-25. The titrant solution was then the proposed electron-transfer site. In the case of cytochrome concentrated by centrifugation above a Vivaspin membrane c550, this electron-transfer site is the exposed heme edge, (Mr cutoff 5000). Protein solutions were degassed, and the while for pseudoazurin it is a histidine coordinating the target cytochrome c peroxidase was placed in the sample buried copper. In both cases, these electron-transfer sites are chamber of the VP-ITC microcalorimeter (Microcal). The the point at which large dipole moments exit the protein syringe was filled with a solution of the probe protein, and surface. This is consistent with a role for the dipole moment successive injections of 10 µL were delivered into the stirred in preorientation during encounter with the negative elec- chamber (after an initial injection of 1 µL). The duration of trostatic field of the cytochrome c peroxidase (1, 12). In this the additions was 20 s, and they were 180 s apart. The paper, we investigate whether the pseudoazurin and the instrument records the heat evolved or absorbed in the sample cytochrome c550 bind at different sites and simultaneously chamber by adjusting a heating circuit to maintain a constant or whether they compete for the same site. temperature, and the data are analyzed with Microcal Origin software, which fits on the basis of iteration within a MATERIALS AND METHODS Marquandt routine. Data were fitted using the one-set-of- Source of the Proteins. Cytochrome c peroxidase was sites model. The value of Kd was used to calculate the ° purified from Paracoccus pantotrophus (LMD 52.44) as standard Gibbs free energy change (∆G ). The standard free described by Goodhew et al. (13). Pseudoazurin was purified energy change and the standard enthalpy change were used ° either from P. pantotrophus LMD 82.5 or the recombinant to calculate a standard entropy change (∆S ). 1 pseudoazurin gene from P. pantotrophus LMD 52.44 was H NMR Spectroscopy. (i) Protein Samples. The protein expressed in Escherichia coli (1). These two sources gave samples (cytochrome c peroxidase, pseudoazurin, and cy- 1 identical proteins (1). Enzyme, cytochrome, and pseudoazurin tochrome c550) were desalted into 10 mM Hepes, 2mM concentrations were determined using extinction coefficients CaCl2, pH 7.5, using a Sephadex G25 column, and concen- for the oxidized forms of 250 mM-1 cm-1 (cytochrome c trated above a Vivaspin membrane (with Mr cutoff 5000). peroxidase, 409 nm), 108 mM-1 cm-1 (cytochrome c550, The binary titrations were carried out using a solution of 410 nm), and 3 mM-1 cm-1 (pseudoazurin, 590 nm). The 0.2 mM cytochrome c peroxidase in 10 mM Hepes, 2 mM latter figure was reevaluated in ref 1. CaCl2, pH 7.5, in 10% D2O with increasing amounts of Analytical Ultracentrifugation. Partial specific volumes of pseudoazurin or cytochrome c550. In the competition experi- the proteins were calculated from the amino acid composition ments, pseudoazurin or cytochrome c550 were added to a and are 0.7318 mL/g (cytochrome c peroxidase), 0.735 mL/g solution containing 0.2 mM peroxidase and either 0.2 mM (cytochrome c550), and 0.7382 mL/g (pseudoazurin) using cytochrome c550 or 0.2 mM pseudoazurin, respectively, until SEDNTERP (based on ref 14). Protein solutions were a 1.5 molar ratio was achieved. equilibrated with the appropriate buffer by molecular exclu- (ii) Data Acquisition. 1D NMR spectra were recorded on sion chromatography on Sephadex G25. The Beckman a Bruker Avance DRX 500 spectrometer at 299 K with a Optima XL-A or XL-I (Beckman, Palo Alto, CA) analytical spectral width of 200 ppm for 32 000 data points and 1024 ultracentrifuges, equipped with scanning absorption optics, scans accumulated. NMR spectra were obtained using were used in all the sedimentation velocity experiments at presaturation of water and processed using xwinnmr provided 45 000 rpm, 25 °C. Sedimentation coefficients (s) were by Bruker. The chemical shifts were referenced to the H2O obtained by scanning at 500 or 530 nm depending on the resonance (4.76 ppm at 299 K). protein concentration in the cell. The extinction coefficients (iii) Data Analysis of Binding in the Presence of Two of the cytochrome c peroxidase at these wavelengths are 17.2 Protein Ligands.
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