Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme.
Ascorbate peroxidase from L. Major (LmAPX) is a functional hybrid between cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX). We utilized point mutagenesis to investigate if a conserved proximal tryptophan residue (Trp208) among Class I peroxidase helps in controlling catalysis. The mutant W208F enzyme had no effect on both apparent dissociation constant of the enzyme-cytochrome c complex and K(m) value for cytochrome c indicating that cytochrome c binding affinity to the enzyme did not alter after mutation. Surprisingly, the mutant was 1000 times less active than the wild type in cytochrome c oxidation without affecting the second order rate constant of compound I formation. Our diode array stopped-flow spectral studies showed that the substrate unbound wild type enzyme reacts with H(2)O(2) to form compound I (compound II type spectrum), which was quite different from that of compound I in W208F mutant as well as horseradish peroxidase (HRP). The spectrum of the compound I in wild type LmAPX showed a red shift from 409 nm to 420 nm with equal intensity, which was broadly similar to those of known Trp radical. In case of compound I for W208F mutant, the peak in the Soret region was decreased in heme intensity at 409 nm and was not shifted to 420 nm suggesting this type of spectrum was similar to that of the known porphyrin pi-cation radical. In case of an enzyme-H(2)O(2)-ascorbate system, the kinetic for formation and decay of compound I and II of a mutant enzyme was almost identical to that of a wild type enzyme. Thus, the results of cytochrome c binding, compound I formation rate and activity assay suggested that Trp208 in LmAPX was essential for electron transfer from cytochrome c to heme ferryl but was not indispensable for ascorbate or guaiacol oxidation.
Yadav
Rajesh K
RK
Division of Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700 032, India.
Dolai
Subhankar
S
Pal
Swati
S
Adak
Subrata
S
D000595:Amino Acid Sequence; D000818:Animals; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D050505:Mutant Proteins; D010649:Phenylalanine; D014364:Tryptophan; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid