title: Electron transfer from HiPIP to the photooxidized tetraheme cytochrome subunit of Allochromatium vinosum reaction center: new insights from site-directed mutagenesis and computational studies.
abstract: The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 +/- 0.2) 10(5) M(-1) s(-1) at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair.
authors.affiliation: Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, Bologna, Italy, Istituto Nazionale per la Fisica della Materia (INFM), UdR di Bologna, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Venturoli
authors.forename: Mahir D
authors.initials: MD
authors.lastname: Mamedov
authors.forename: Sheref S
authors.initials: SS
authors.lastname: Mansy
authors.forename: Francesco
authors.initials: F
authors.lastname: Musiani
authors.forename: Massimo
authors.initials: M
authors.lastname: Strocchi
authors.forename: Francesco
authors.initials: F
authors.lastname: Francia
authors.forename: Alexey Yu
authors.initials: AY
authors.lastname: Semenov
authors.forename: James A
authors.initials: JA
authors.lastname: Cowan
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D019295:Computational Biology / Q000379:methods; D017124:Conserved Sequence / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry; D004579:Electron Transport / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D020560:Proteobacteria / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D021122:Protein Subunits; C007943:high potential iron-sulfur protein; D010649:Phenylalanine; C017165:cytochrome c(3)