title: Nitrate as a probe of cytochrome c surface: crystallographic identification of crucial "hot spots" for protein-protein recognition.
abstract: The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred.
authors.affiliation: Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: De March
authors.affiliation: Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Demitri
authors.affiliation: Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: De Zorzi
authors.affiliation: Dept. Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Angela
authors.initials: A
authors.lastname: Casini
authors.affiliation: Department of Chemistry and Industrial Chemistry, University of Pisa, I-56126 Pisa, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Gabbiani
authors.affiliation: Department of Chemistry, University of Florence, I-50019 Sesto Fiorentino (FI), Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Guerri
authors.affiliation: Department of Chemistry, University of Florence, I-50019 Sesto Fiorentino (FI), Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Messori
authors.affiliation: Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L. Giorgeri 1, 34127 Trieste, Italy. Electronic address: sgeremia@units.it.
authors.forename: Silvano
authors.initials: S
authors.lastname: Geremia
mesh_terms: D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D045304:Cytochromes c / Q000737:chemistry*; D006736:Horses; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009566:Nitrates / Q000737:chemistry*; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D013499:Surface Properties
chemical_list: D009566:Nitrates; D045304:Cytochromes c
keywords: Cytochrome c; Electron transfer complexes; Electrostatic surface; Redox protein; Reduction potential