Open AccessThe surface‐charge asymmetry and dimerisation of cytochrome c 550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase
Author(s) -
Pettigrew G. W.,
Gilmour R.,
Goodhew C. F.,
Hunter D. J. B.,
Devreese B.,
Van Beeumen J.,
Costa C.,
Prazeres S.,
Krippahl L.,
Palma P. N.,
Moura I.,
Moura J. J. G.
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2580559.x
Subject(s) - paracoccus denitrificans , chemistry , dimer , cytochrome c , cytochrome , cytochrome c peroxidase , stereochemistry , crystallography , monomer , molecular mass , organic chemistry , biochemistry , enzyme , mitochondrion , polymer
The implications of the dimeric state of cytochrome c 550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c 550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X‐ray structure of the latter, a structure for the cytochrome c 550 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 kDa) equilibrium that can also be detected by 1 H‐NMR spectroscopy. The apparent 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‐linking studies, which showed that a cross‐linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular‐exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1 H‐NMR spectroscopy shows that it is the monomer that binds.