Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase

To enhance the oxidation activity for luminol in horseradish peroxidase (HR P), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold h igher oxidation activity for luminol than native HRP. Spectroscopic studies by H-1 NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of l uminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, whic h are located near the 7-propionate and 8-methyl groups of the heme. are in volved in the specific binding to luminol. The positively charged Lys and n egatively charged Glu were introduced into the corresponding positions of S er35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme su bstituents and heme environmental residues in the H-1 NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induc ed interactions with luminol in the vicinity of the heme. The catalytic eff iciencies (V-max/K-m) for luminol oxidation of the S35K and S35K/Q176E muta nts were 1.5- and 2-fold improved, whereas that of the Q176E mutant was sli ghtly depressed. The increase in luminol activity of the S35K and S35K/Q176 E mutants was rather small but significant, suggesting that the electrostat ic interactions between the positive charge of Lys35 and the negative charg e of luminol can contribute to the effective binding for the luminol oxidat ion. On the other hand, the negatively charged residue would not be so cruc ial for the luminol oxidation. The absence of drastic improvement in the lu minol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol a s observed for ARP.