Characterization of Polyethylene Glycolated Horseradish Peroxidase in Organic Solvents: Generation and Stabilization of Transient Catalytic Intermediates at Low Temperature

Polyethylene glycolated horseradish peroxidase (PEG-HRP) can catalyze one- and two-electron oxidation reactions in organic solvents as well as in aqueous buffer. Even though the oxidation of guaiacol in benzene and chlorobenzene is 5 orders of magnitude slower than in phosphate buffer, compounds I and II are involved in the catalytic cycle in organic media. Factor analysis and global fittings of rapid scan data set reveal that the formation of compound I of PEG-HRP in organic media consists of two steps (the first fast and the second slow) and suggest the involvement of a H2O2−HRP complex in the catalytic cycle. The labile precursor of compound I is stabilized when PEG-HRP reacts with hydrogen peroxide in chlorobenzene at −20 °C. The absorption spectrum of the precursor does not exhibit the features of hyperporphyrin spectrum but has a normal Soret as previously observed in R38L HRP. More importantly, compound I of PEG-HRP can be maintained for more than an hour at −20 °C in chlorobenzene

Mechanisms of Sulfoxidation Catalyzed by High-Valent Intermediates of Heme Enzymes: Electron-Transfer vs Oxygen-Transfer Mechanism

Mechanisms of sulfoxidation catalyzed by high-valent intermediates of heme enzymes have been investigated by direct observation of sulfide-induced reduction of three different compound I species including HRP (horseradish peroxidase), the His64Ser myoglobin (Mb) mutant, and OFeIVTMP+• (1) (TMP = 5,10,15,20-tetramesitylporphyrin dianion). The reaction of thioanisole and compound I of HRP (10 μM, pH 7.0, 298 K) gives the resting state of HRP with accumulation of compound II as an intermediate. The yield of sulfoxide by a stoichiometric reaction of HRP compound I with thioanisole was only 25% ± 5%. On the other hand, the same sulfoxidation by both 1 and His64Ser Mb compound I exclusively exhibited a two-electron process, resulting in quantitative formation of sulfoxide. When 1,5-dithiacyclooctane (DTCO) is employed as a substrate, the reaction of His64Ser Mb compound I with DTCO exhibits rapid formation of compound II, which decays to the ferric state due to the low oxidation potential of DTCO. The observed rate constants (log kobs) of the reactions of 1 and compounds I of HRP and His64Ser Mb with a series of p-substituted thioanisoles correlate with the one-electron oxidation potentials (E0ox) of the sulfides. A comparison of these correlations with the established correlation between log kobs and E0ox for the corresponding electron-transfer reactions of substituted N,N-dimethylanilines has revealed that the sulfoxidation reactions of compound I of HRP with the sulfides proceed via electron transfer while the sulfoxidations catalyzed by 1 and compound I of His64Ser Mb occur via direct oxygen transfer

Effects of the Arrangement of a Distal Catalytic Residue on Regioselectivity and Reactivity in the Coupled Oxidation of Sperm Whale Myoglobin Mutants

The coupled oxidations of sperm whale myoglobin (Mb) mutants are performed to examine active site residues controlling the regiospecific heme degradation. HPLC analysis of biliverdin isomers shows that L29H/H64L Mb almost exclusively gives biliverdin IXγ, although H64L and wild-type Mb mainly afford the α-isomer. Relocation of the distal histidine at the 43 and 107 positions increases the amount of γ-isomer to 44 and 22%, respectively. Interestingly, the increase in the ratio of γ-isomer is also observed by a single replacement of either His-64 with Asp or Phe-43 with Trp. It appears that the polarity of the active site as well as hydrogen bonding between oxygen molecule bound to the heme iron and His or Trp is important in controlling the regioselectivity. The results of coupled oxidation kinetics, autoxidation kinetics, and redox potential of the Fe3+/Fe2+ couple are discussed with regard to their implications for the active site and mechanism of heme oxygenase