CO recombination in cytochrome c peroxidase: effect of the local heme environment on CO binding explored through site-directed mutagenesis.
CO recombination to the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis. The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly. At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0. This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization. The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1). Kinetic forms analogous to forms A and B were observed in all of the mutants examined. A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH. Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants. Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination. The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however. The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.
Miller
M A
MA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Coletta
M
M
Mauro
J M
JM
Putnam
L D
LD
Farnum
M F
MF
Kraut
J
J
Traylor
T G
TG
D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism*; D003001:Cloning, Molecular; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D006418:Heme / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009154:Mutation; D010544:Peroxidases / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase