Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners.
The transient electron transfer (ET) interactions between cytochrome c1 of the bc1-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c552 and cytochrome c550 have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c1 were generated and used together with a soluble cytochrome c552 module as a model system for interprotein ET reactions. Both c1 fragments lack the membrane anchor; the c1 core fragment (c1CF) consists of only the hydrophilic heme-carrying domain, whereas the c1 acidic fragment (c1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c1 reveals fast bimolecular rate constants (10(7) to 10(8) M(-1) s(-1)) for the ET reaction with its physiological substrates c552 and c550, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c1-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c550 over c552 reacting with cyochrome c1 was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.
Janzon
Julia
J
Molecular Genetics Group, Institute of Biochemistry, Biocentre J. W. Goethe-University Frankfurt/Main, Germany.
Eichhorn
Anna Carina
AC
Ludwig
Bernd
B
Malatesta
Francesco
F
D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000378:metabolism*; D003575:Cytochromes c1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004058:Diffusion; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis
D003574:Cytochrome c Group; D010446:Peptide Fragments; D003575:Cytochromes c1; C022302:cytochrome C-552; C031732:cytochrome C-550; D014450:Electron Transport Complex III