To clarify how the location of distal histidine affects the activation process of H 2 O 2 by heme proteins, we have characterized reactions with H 2 O 2 for the L29H/ H64L and F43H/H64L mutants of sperm whale myoglobin (Mb), designed to locate the histidine farther from the heme iron. Whereas the L29H/H64L double substitution retarded the reaction with H 2 O 2 , an 11-fold rate increase versus wild-type Mb was observed for the F43H/ H64L mutant. The V max values for 1-electron oxidations by the myoglobins correlate well with the varied reactivities with H 2 O 2 . The functions of the distal histidine as a general acid-base catalyst were examined based on the reactions with cumene hydroperoxide and cyanide, and only the histidine in F43H/H64L Mb was suggested to facilitate heterolysis of the peroxide bond. The x-ray crystal structures of the mutants confirmed that the distal histidines in F43H/H64L Mb and peroxidase are similar in distance from the heme iron, whereas the distal histidine in L29H/H64L Mb is located too far to enhance heterolysis. Our results indicate that the proper positioning of the distal histidine is essential for the activation of H 2 O 2 by heme enzymes.Peroxidase is a heme enzyme that catalyzes 1-electron oxidations of a variety of substrates (1, 2). The ferric enzyme is oxidized by H 2 O 2 to yield a ferryl porphyrin cation radical (Fe IV ϭO Por . ϩ ) known as compound I in the first step of the catalytic cycle (3). Compound I is reduced to the ferric state through a ferryl species (Fe IV ϭO Por), so-called compound II, by two sequential 1-electron oxidations of substrates. The invariant histidine in the distal heme pocket (1-6) is a critical residue for peroxidases, and its replacement by aliphatic residues retards compound I formation by 5ϳ6 orders of magnitude (7-9). As shown in Scheme I, the distal histidine is believed to function (i) as a general base to accelerate binding of H 2 O 2 to the ferric heme iron by deprotonating the peroxide and (ii) as a general acid to facilitate the heterolytic cleavage of the O-O bond of a plausible Fe III ⅐OOH complex by protonating the terminal oxygen atom (10). The charge separation in heterolysis is suggested to be also enhanced by a positively charged distal arginine (Scheme I), whose substitution results in 2 orders of magnitude slower formation of compound I (10 -12).Myoglobin (Mb), 1 a carrier of molecular oxygen, similarly possesses a distal histidine (His-64) in the heme pocket ( Fig. 1 might be partly due to the malfunction of the distal histidine as a general acid-base catalyst and the absence of the distal arginine. Whereas the distal histidine in peroxidase is suggested to raise the basicity of imidazole by a hydrogen bond with the adjacent asparagine (20, 21), the absence of the hydrogen bond in Mb (13, 14) is indicative of less basicity of its distal histidine. Furthermore, wild-type Mb cleaves the O-O bond of the heme-bound peroxide not only heterolytically, but also homolytically to give Mb-II and a hydroxy radical as shown in Scheme...

A His64 f Asp mutant of sperm whale myoglobin (Mb), H64D Mb, has been prepared to mimic the active site of chloroperoxidase from the marine fungus Caldariomyces fumago, in which distal glutamic acid is suggested to enhance compound I formation by H 2 O 2 . The H64D mutant allows us to see the accumulation of compound I in the reaction of Mb with H 2 O 2 for the first time. The successful observation of compound I is due to at least 50-fold improvement in the formation rate of compound I as well as its stabilization upon the His64 f Asp replacement. Catalytic activity of wild-type Mb and a series of His64 Mb mutants (H64A, H64S, H64L, and H64D Mb) are examined for one-electron oxidation and oxygenation by using H 2 O 2 as an oxidant. The H64D mutant is the best catalyst among the myoglobins and shows 50-70-fold and 600-800-fold higher activity than the wild type in the one-electron oxidations and peroxygenations, respectively. The origin of the varied activity upon the mutations is discussed on the basis of the formation rate and stability of compound I.

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 k obs) 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 (E 0 ox) of the sulfides. A comparison of these correlations with the established correlation between log k obs and E 0 ox 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.

Myoglobin (Mb) catalyzes various two-electron oxidations; however, ferryl porphyrin cation radical equivalent to peroxidase compound I has not been identified yet. Distal histidine mutants of sperm whale Mb (His-64 3 Ala, Ser, and Leu) afford an apparent intermediate followed by the formation of a ferryl heme (Mb-II) in the reaction with m-chloroperbenzoic acid. Because the intermediate exhibits characteristic absorption spectrum of compound I and bears two electron oxidizing equivalents above the ferric state, we have assigned the species as compound I of myoglobin (Mb-I). Although we have recently observed compound I of the F43H/H64L Mb mutant, F43H and wild type Mb react with m-chloroperbenzoic acid to give Mb-II without any accumulation of Mb-I. The results unambiguously indicate that His-64 plays a key role in destabilizing wild type Mb-I. Furthermore, Mb-I is found to be capable of performing two-electron oxidation of styrene, thioanisole, and H 2 O 2 .The key intermediate in the catalytic cycles of heme-containing peroxidase and catalase are ferryl porphyrin cation radicals (Fe IV ϭ O Por ϩ ⅐ ) called compound I formed by the reaction of the resting ferric enzymes with peroxide (1, 2). By the two sequential one-electron transfers from substrates, compound I is reduced back to the ferric state via a ferryl heme (Fe IV ϭ O Por) known as compound II. The two-electron oxidation that is often associated with the ferryl oxygen transfer to substrates also takes place by compound I. The reactive species responsible for the oxygenation by cytochrome P-450, heme-containing monooxygenase is assumed to be compound I (3, 4).Myoglobin (Mb), 1 a carrier of molecular oxygen, can catalyze H 2 O 2 -dependent two-electron oxidations including styrene epoxidation (5-8); however, the reactions of Mb with peroxides are known to yield a ferryl heme (Mb-II) coupled to a transient protein radical in stead of a ferryl porphyrin cation radical (Mb-I) (9). The epoxidation by wild type Mb in the presence of O incorporation into the epoxide, and oxygen in the epoxide was derived primarily (80%) from molecular oxygen (5, 6). The incorporation of the molecular oxygen has been attributed to the co-oxidation by the protein-peroxy radical, which is formed by the reaction of molecular oxygen and the protein radical (Scheme I). Thus, Mb-I appears to be a branch to co-oxidation versus ferryl oxygen transfer mechanism.The Mb mutants in which distal histidine (His-64, E7) is replaced by Leu or Val (Fig. 1) showed drastic increase in the ratio of ferryl oxygen transfer even though the Mb-I as well as Mb-II of those mutants has not been observed when H 2 O 2 is used as an oxidant (6, 10, 11). We previously designed a F43H/ H64L mutant to mimic the active site of peroxidases because the distal histidine of peroxidases functions as a general acidbase catalyst in the formation of compound I (12). Compound I of the F43H/H64L mutant was not detected in the presence of H 2 O 2 due to the enhanced catalase activity of the mutant, whereas the reaction of...