Monooxygenation of an Aromatic Ring by F43W/H64D/V68I Myoglobin Mutant and Hydrogen Peroxide

Abstract

Myoglobin (Mb)1 is a small (17 kDa), well characterized heme protein that is often used as a model system for other heme proteins and the reactions they catalyze. In addition to its native function as an oxygen carrier, Mb has been engineered to efficiently perform peroxidase, catalase, and peroxygenase (sulfoxidation and epoxidation) activities (1-4). Scheme 1 shows three major alternate pathways for the reaction with peroxide. The latest novel function to be proposed for Mb is cytochrome P450-type aromatic carbon hydroxylation. Although hydroxylation by P450s has been extensively studied (5-7), the mechanism is still not fully understood (6,8). Input from new model systems could provide additional insight. P450 enzymes capture their substrates near the heme via specific interactions (9 -11), which Mb cannot do. A Trp was, thus, engineered in the heme pocket of Mb to model P450 hydroxylation of aromatic compounds ( Fig. 1) (12, 13). The monooxygenation product has not previously been observed or isolated due to rapid subsequent oxidation steps.The mechanism of hydroxylation by P450s is proving to be quite complex and may even vary among various P450s (7). Some of the proposed mechanisms involve 1) an epoxide intermediate for the aromatic hydroxylation, 2) a concerted direct insertion of oxygen, 3) a non-concerted sequential insertion involving hydrogen abstraction and oxygen rebound, or 4) a more simple non-concerted radical mechanism (6 -8, 14). Recently it has been found that these mechanisms may not be entirely valid, and the actual mechanism may involve two electrophilic oxidants and/or two spin states of the iron oxo species (6,8,14). Although P450cam has been shown to lose its hydroxylation activity when an imidizole is substituted for the axial thiolate ligand (15, 16), in contrast, chloroperoxidase retains its chlorination, peroxidation, epoxidation, and catalase activities when its thiolate ligand is mutated to histidine (17). Also, studies with iron porphyrin compounds have demonstrated that imidazole ligation can replace the thiolate in P450 type reactions under certain conditions (18). Therefore, because * This work was supported by Grants-in-Aid for Scientific Research 14209019 (to Y. W). and 13740384 (to T. U.) and by the 21st Century COE program "Establishment of COE on Materials Science: Elucidation and Creation of Molecular Functions" of Nagoya University (to T. D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** Present address: National Institute of Advanced Industrial Science and Technology Research Institute of Genome-based Biofactory, 2-17 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan.ʈʈ To whom correspondence should be addressed. Tel.: 81-52-789-3049; Fax: 81-52-789-2953; E-mail: yoshi@nucc.cc.nagoya-u.ac.jp. 1 The abbreviations used are: Mb, myoglobin; Mb WDI, Mb F43W/ H64D/V68I mutant; Mb WL, Mb ...

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

“…Mb is readily autoxidized to metMb in an aerobic environment (reviewed in [9]), and enzymatic reduction of myoglobin to the reduced, Fe(II) state was reported over twenty years ago [10] though the subsequent literature has produced inconsistent results regarding the nature of this enzymatic system (reviewed in [11]). The literature concerning the oxidation-reduction properties and electron transfer kinetics of myoglobin is extensive (a review of the older literature is provided in [12]) and in more recent years has emphasized the use of myoglobin as (i) a model for studies of intramolecular electron transfer (e.g., [13,14]), (ii) a participant in protein-protein electron transfer reactions (e.g., [15][16][17][18]), a model for ligand binding (e.g., [19][20][21][22][23]) and (iv) a protein scaffold for genetic engineering of new functionalities (e.g., [19,[24][25][26][27][28]). …”

Generation of New Artificial Metalloproteins by Cofactor Modification of Native Hemoproteins

“…The axial ligand of myoglobin is the imidazole of histidine, and no clear substrate binding site exists. It is well known that myoglobin is not capable of promotion of hydroxylation of an inert alkane substrate via CÀH bond activation (except one example) [26] In addition, the highly oxidized intermediate known as compound I (an oxoferryl porphyrin p cationic species) is not detectable in native myoglobin.…”

Advances in Engineered Hemoproteins that Promote Biocatalysis

“…A series of reports have described the engineering of the myoglobin active site to induce peroxygenase (sulfoxidation and epoxidation) and peroxidase reactivity [4,9,[44][45][46][47][48]. Many mutants have been described, and some have showed increased reactivity.…”

“…In its native form, peroxidative reactivity of substrates is not very efficient even though oxidative intermediates are presumably formed from the reaction with peroxides. Lack of reactivity of the native form of myoglobin has been examined and many research studies have concluded that the active site of myoglobin lacks a substrate-binding site, which would prohibit oxidative intermediates to productively react with substrates [4,8,24,25,[44][45][46][47][48]. To this end, many studies have employed the use of myoglobin mutants to affect a change of reactivity by (a) opening up the active site to afford substrate binding or (b) changing amino acids at the active site to mimic structural aspects of other enzymes and their functions.…”

“…The distal histidine residue in myoglobin is in close proximity to the heme and it serves as a hydrogen bond acceptor. The H64D mutant of myoglobin is shown to react with m-chloroperbenzoic acid (mCPBA) to afford a compound I species that promotes peroxidase activity showing reactivity towards guaiacol (2-methoxyphenol) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which are common target substrates to assay for peroxidase activity [44,47]. .…”