Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome.
According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.
Pettigrew
Graham W
GW
Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, UK.
Pauleta
Sofia R
SR
Goodhew
Celia F
CF
Cooper
Alan
A
Nutley
Margaret
M
Jumel
Kornelia
K
Harding
Stephen E
SE
Costa
Cristina
C
Krippahl
Ludwig
L
Moura
Isabel
I
Moura
Jose
J
D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D003198:Computer Simulation; D003574:Cytochrome c Group / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D004579:Electron Transport; D006736:Horses; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011522:Protons; D012996:Solutions; D014461:Ultracentrifugation
D003574:Cytochrome c Group; D009097:Multienzyme Complexes; D011522:Protons; D012996:Solutions; D045304:Cytochromes c; C031732:cytochrome C-550; D003578:Cytochrome-c Peroxidase