The surface-charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans - implications for the interaction with cytochrome c peroxidase

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons r equired to restore the active enzyme during catalysis have been investigate d. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans s train LMD 52.44 was determined and showed 21 differences from that of strai n LMD 22.21. Based on the X-ray structure of the latter, a structure for th e cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kD a) equilibrium that can also be detected by H-1-NMR spectroscopy. The appar ent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-li nking studies, which showed that a cross-linked species of mass 30 kDa on S DS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatog raphy. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the mono mer dipoles lay almost antiparallel to each other. The resultant dipole mom ent of the dimer is therefore small. Although this finding calls into quest ion the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of comple x formation with cytochrome c peroxidase as studied by H-1-NMR spectroscopy shows that it is the monomer that binds.