Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2).
Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.
Koh
Moonjoo
M
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.
Meyer
Terry E
TE
De Smet
Lina
L
Van Beeumen
Jozef J
JJ
Cusanovich
Michael A
MA
D002118:Calcium / Q000378:metabolism; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D045362:Cytochromes c2; D004579:Electron Transport; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D013997:Time Factors
D003574:Cytochrome c Group; D007477:Ions; D006418:Heme; D045362:Cytochromes c2; D003578:Cytochrome-c Peroxidase; D002118:Calcium