ATR-FTIR Redox Difference Spectroscopy of Yarrowia lipolytica and Bovine Complex I

• Published in: Biochemistry (Easton) Vol. 45; no. 17; pp. 5458 - 5467
• Main Authors: Marshall, Douglas, Fisher, Nicholas, Grigic, Ljuban, Zickermann, Volker, Brandt, Ulrich, Shannon, Richard J, Hirst, Judy, Lawrence, Rebecca, Rich, Peter R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02-05-2006
• Subjects: Animals; Cattle; Deuterium Exchange Measurement; Electron Transport Complex I - chemistry; Flavin Mononucleotide - chemistry; Hydrogen-Ion Concentration; Myocardium - enzymology; Nitrogen Isotopes; Oxidation-Reduction; Spectroscopy, Fourier Transform Infrared - methods; Ubiquinone - chemistry; Yarrowia - enzymology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi052561e

ATR-FTIR spectroscopy in combination with electrochemistry has been applied to the redox centers of Yarrowia lipolytica complex I. The redox spectra show broad similarities with previously published data on Escherichia coli complex I and with new data here on bovine complex I. The spectra are dominated by amide I/II protein backbone changes. Comparisons with redox IR spectra of small model ferredoxins demonstrate that these amide I/II changes arise primarily from characteristic structural changes local to the iron−sulfur centers, rather than from global structural alterations as has been suggested previously. Bands arising from the substrate ubiquinone were evident, as was a characteristic 1405 cm-1 band of the reduced form of the FMN cofactor. Other signals are likely to arise from perturbations or protonation changes of a carboxylic amino acid, histidine, and possibly several other specific amino acids. Redox difference spectra of center N2, together with substrate ubiquinone, were isolated from those of the other iron−sulfur centers by selective redox potentiometry. Its redox-linked amide I/II changes were typical of those in other 4Fe-4S iron sulfur proteins. Contrary to published data on bacterial complex I, no center N2 redox-linked protonation changes of carboxylic amino acids or tyrosine were evident, and other residues that could provide its redox-linked protonation site are discussed. Features of the substrate ubiquinone associated with the center N2 spectrum were particularly clear, with firm assignments possible for bands from both oxidized and reduced forms. This is the first report of IR properties of ubiquinone in complex I, and the data could be used to estimate a stoichiometry of 0.2−0.4 per complex I.