Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c(1) of the bc(1)-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c(552) and cytochrome c(550) have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c(1) were generated and used together with a soluble cytochrome c(552) module as a model system for interprotein ET reactions. Both c(1) fragments lack the membrane anchor; the c(1) core fragment (c(1CF)) consists of only the hydrophilic heme-carrying domain, whereas the c1 acidic fragment (c(1AF)) additionally contains the acidic domain unique top 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 c(1) reveals fast bimolecular rate constants (10(7) to 10(1) M-1 s(-1)) for the ET reaction with its physiological substrates c(552) and c(550), 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 c(1)-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c(550) over c(552) reacting with cyochrome c(1) was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans. (C) 2008 Elsevier B.V. All rights reserved.