title: Orthogonal translation meets electron transfer: in vivo labeling of cytochrome c for probing local electric fields.
abstract: Cytochrome c (cyt c), a redox protein involved in diverse fundamental biological processes, is among the most traditional model proteins for analyzing biological electron transfer and protein dynamics both in solution and at membranes. Studying the role of electric fields in energy transduction mediated by cyt c relies upon appropriate reporter groups. Up to now these had to be introduced into cyt c by in vitro chemical modification. Here, we have overcome this restriction by incorporating the noncanonical amino acid p-cyanophenylalanine (pCNF) into cyt c in vivo. UV and CD spectroscopy indicate preservation of the overall protein fold, stability, and heme coordination, whereas a small shift of the redox potential was observed by cyclic voltammetry. The C≡N stretching mode of the incorporated pCNF detected in the IR spectra reveals a surprising difference, which is related to the oxidation state of the heme iron, thus indicating high sensitivity to changes in the electrostatics of cyt c.
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623 Berlin (Germany); Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin (Germany).
authors.forename: Jan
authors.initials: J
authors.lastname: Völler
authors.forename: Hernan
authors.initials: H
authors.lastname: Biava
authors.forename: Beate
authors.initials: B
authors.lastname: Koksch
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D008958:Models, Molecular; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D010649:Phenylalanine / Q000031:analogs & derivatives
chemical_list: D000409:Alanine; D045304:Cytochromes c; D009570:Nitriles; C530634:p-cyanophenylalanine; D010649:Phenylalanine
keywords: cyanophenylalanine; cytochromes; infrared spectroscopy; non-canonical amino acids; redox chemistry; stop codon suppression