Histidine 52 is a critical residue for rapid formation of cytochrome c peroxidase compound I.
The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase [CcP(MI)], in which the conserved distal His (His-52) is replaced with Leu. The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength. The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively. This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions. Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation. Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules. The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent. In nitrate-containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pKA = 4.5. Protonation of the group caused a loss of reactivity with peroxide. This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pKA = 5.4.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry, Northern Illinois University, DeKalb 60115.
J E
JE
Erman
L B
LB
Vitello
M A
MA
Miller
A
A
Shaw
K A
KA
Brown
J
J
Kraut
D003461:Crystallography; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
D005656:Fungal Proteins; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase