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

Abstract

The implications of the dimeric state of cytochrome c550 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 c550 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 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 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.Keywords : cytochrome; peroxidase ; electron transfer; electrostatic ; dimerisation.Paracoccus denitrificans cytochrome c550 is the electron donor for Paracoccus cytochrome c peroxidase (CCP). We have used these two redox proteins as a model system, with which to investigate and understand electron transfer between proteins. They represent a useful parallel system to the well-studied yeast cytochrome c and yeast CCP [1,2] in that the cytochrome c donors are structurally similar but the peroxidases are quite distinct.Like the yeast peroxidase, the Paracoccus enzyme requires two electrons to restore the active form after oxidation by hydrogen peroxide. In the yeast case, these are probably delivered singly by two ferrocytochrome c molecules sequentially encountering the peroxidase surface. The Paracoccus enzyme contains two haems rather than one, and in separate domains of the protein [3]. One haem is believed to perform an electrontransferring function and the other, a peroxidatic function. With two domains, each carrying an oxidising centre in the intermedi-

Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri

“…In contrast, with horse heart cytochrome c as donor, the fall in activity with increasing ionic strength is indicative of the interaction of two oppositely charged molecules [26], as seen for Pa. denitrificans and its electron donors. Interestingly, however, we do not observe the initial increase in activity with ionic strength in the low ionic strength region that is observed for the Pa. denitrificans enzyme [24]. In the latter this was interpreted as due to the formation of too tight a binding at very low ionic strength, which either prevents the mobility within the encounter complex needed for fast electron transfer [27] or leads to slow dissociation and inhibition of turnover.…”

“…The effect of the ionic strength on the enzymatic activity of CCP with cytochrome c 551 as electron donor was different from that obtained with the horse heart cytochrome c and also from the effect of ionic strength on the reactivity of Pa. denitrificans CCP [24]. With cytochrome c 551 , the catalytic center activity increases with ionic strength (Fig.…”

The Structure of an Electron Transfer Complex Containing a Cytochrome c and a Peroxidase

“…Although this cytochrome is present in a monomer:dimer equilibrium in solution, we have shown that it is the monomer that binds to the peroxidase (16). Cytochrome c 550 is a close relative of mitochondrial cytochrome c and has a pronounced charge asymmetry with a positive front surface surrounding an exposed heme edge at which its proposed attachment to a negative surface of the peroxidase occurs (16,17). The model of Fig.…”

“…This sequence was mutated in Sybyl (Tripos Associates) to correspond to the cytochrome c 550 from strain LMD 52.44 (21 differences, see Ref. 16). The CCP from P. aeruginosa was mutated in Sybyl to correspond to the protein from P. denitrificans strain 52.44 (61% identity).…”

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

“…Pap ccp is one such periplasmic enzyme [3] which can accept electrons from either a cytochrome c 550 or a pseudoazurin [45,46]. The mid-point potential (E m,7 ) of the E haem (226 mV) [18] is close to that of the cytochrome c 550 (265 mV) and the pseudoazurin (250 mV) (Pauleta and Pettigrew, unpublished results).…”