title: The formation of protein complexes between ferricytochrome b5 and ferricytochrome c studied using high-resolution 1H-NMR spectroscopy.
abstract: The association of the tryptic fragment of bovine microsomal cytochrome b5 with cytochrome c has been studied by one- and two-dimensional 1H-NMR spectroscopy. The association of cytochromes to form protein complexes is apparent from the increase in linewidths for resonances of ferricytochrome b5 as well as small perturbations in their chemical shifts that occur upon increasing the cytochrome c/b5 molar ratio. The changes in the chemical shifts of hyperfine shifted resonances of ferricytochrome b5 with increasing ratios of ferricytochrome c indicate the formation of binary 1:1 complexes and ternary 1:2 complexes. Similarly, titrations of the linewidth of resolved resonances of ferricytochrome b5 are consistent with stoichiometries of 1:1 and 1:2 for complexes formed between cytochromes b5 and c. Surprisingly, in the 1:1 complex, mobility is shown to be a function of ionic strength. Two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) of the binary complex formed between ferricytochrome b5 and c indicate that the positions of many resonances attributable to amino acids are unaltered by protein association, although distinctive chemical shift changes are detected in the alpha-CH of the haem C17 propionate. The protein complex detected by NMR is discussed with respect to the model for the binary complex proposed by Salemme and possible mechanisms of electron transfer.
authors.affiliation: Department of Biochemistry, University of Oxford, England.
authors.forename: D
authors.initials: D
authors.lastname: Whitford
authors.forename: D W
authors.initials: DW
authors.lastname: Concar
authors.forename: N C
authors.initials: NC
authors.lastname: Veitch
authors.forename: R J
authors.initials: RJ
authors.lastname: Williams
mesh_terms: D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry*; D015786:Cytochromes b5 / Q000737:chemistry*; D004579:Electron Transport; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008862:Microsomes, Liver / Q000737:chemistry; D014357:Trypsin
chemical_list: D003574:Cytochrome c Group; D015786:Cytochromes b5; D014357:Trypsin