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Unique Coupling of Mono- and Dioxygenase Chemistries in a Single Active Site Promotes Heme Degradation

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Unique Coupling of Mono- and Dioxygenase Chemistries in a Single Active Site Promotes Heme Degradation Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation Toshitaka Matsuia,1, Shusuke Nambua, Celia W. Gouldingb,c, Satoshi Takahashia, Hiroshi Fujiid, and Masao Ikeda-Saitoa,1 aInstitute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba, Sendai 980-8577, Japan; bDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697; cDepartment of Pharmaceutical Sciences, University of California, Irvine, CA 92697; and dDepartment of Chemistry, Graduate School of Humanities and Sciences, Nara Women’s University, Kitauoyanishi, Nara 630-8506, Japan Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved February 26, 2016 (received for review November 26, 2015) − + Bacterial pathogens must acquire host iron for survival and colo- RH + O2 + 2e + 2H → ROH + H2O, [1] nization. Because free iron is restricted in the host, numerous pathogens have evolved to overcome this limitation by using a R + O → RðOÞ . [2] family of monooxygenases that mediate the oxidative cleavage of 2 2 heme into biliverdin, carbon monoxide, and iron. However, the Biological heme degradation proceeds through a unique self- etiological agent of tuberculosis, Mycobacterium tuberculosis, ac- oxidation mechanism where the substrate heme activates O complishes this task without generating carbon monoxide, which 2 potentially induces its latent state. Here we show that this unusual molecules. The canonical enzyme, heme oxygenase (HO), de- heme degradation reaction proceeds through sequential mono- grades heme into ferrous iron, carbon monoxide (CO), and bil- and dioxygenation events within the single active center of MhuD, iverdin by three successive monooxygenation reactions (Fig. 1A) a mechanism unparalleled in enzyme catalysis. A key intermediate (9, 10). This mechanism, which passes through both hydroxy- of the MhuD reaction is found to be meso-hydroxyheme, which heme and verdoheme intermediates, has been considered the “ ” reacts with O2 at an unusual position to completely suppress its gold standard of heme degradation even for recently identified monooxygenation but to allow ring cleavage through dioxyge- noncanonical HO-type enzymes (11–13). We have discovered, nation. This mechanistic change, possibly due to heavy steric however, that MhuD cleaves the porphyrin ring without releasing deformation of hydroxyheme, rationally explains the unique CO, which is a clear indication of a distinct mechanism, without BIOCHEMISTRY heme catabolites of MhuD. Coexistence of mechanistically dis- producing the verdoheme intermediate (14). MhuD products tinct functions is a previously unidentified strategy to expand termed “mycobilins” (mycobilin-a and -b in Fig. 1B) retain the the physiological outcome of enzymes, and may be applied to α-meso carbon atom at the cleavage site as a formyl group with engineer unique biocatalysts. an extra oxidation at either the β-orδ-meso position. MhuD has heme degradation | monooxygenation | dioxygenation | a single active site where heme is bound in an unusually distorted conformation, best described as ruffled (Fig. 1C and Fig. S1) (15, Mycobacterium tuberculosis | MhuD CHEMISTRY 16). The ruffling is expected to drastically modulate the heme- dependent O activation to initiate this unique mechanism (14). xygenases catalyze substrate oxidation with incorporation 2 Similar heme ruffling is observed for IsdG and IsdI (heme- Oof one or two oxygen atoms from O2. The monooxygena- tion and dioxygenation reactions have little in common at the degrading components of the iron regulated surface determinant mechanistic levels and require different protein scaffolds, so that system), MhuD homologs in Staphylococcus aureus,whichliberate no enzyme is thought to catalyze the two distinct oxygenation formaldehyde upon heme degradation instead of CO (17, 18). modes in a single active site. Monooxygenases activate O by 2 Significance using two electrons and two protons, which in total is equivalent to hydrogen peroxide (H2O2), and results in the release of one Oxygenases are classified into two groups, mono- and dioxy- oxygen atom as a water molecule (Eq. 1). Efficient proton and genases, which, respectively, catalyze insertion of one and two electron transfer machinery is equipped within the active sites oxygen atoms of O2 into substrates. These two modes of ox- of monooxygenases including cytochrome P450, where O2 is ygenation proceed through distinct reaction mechanisms and reductively activated on the heme iron to form a high-valent oxo- require totally different active site structures. For more than 60 species responsible for substrate monooxygenation (1, 2). In years, oxygenase enzymes thus far have been found to cata- contrast, dioxygenases directly add two oxygen atoms of O2 into lyze either a mono- or dioxygenation reaction. This study has substrates without consuming electrons and protons (Eq. 2). broken this paradigm as it clearly demonstrates that the heme- Dioxygenase active sites are generally hydrophobic as observed degrading enzyme from Mycobacterium tuberculosis, MhuD, catalyzes successive mono- and dioxygenation reactions within for tryptophan and indoleamine 2,3-dioxygenases, where O2 binds ferrous heme iron akin to cytochrome P450, but perform distinct its single active site. This unprecedented bifunctionality of the single active center rationally explains the unique heme ca- chemistry (3–5). Prostaglandin-endoperoxide synthase, also known tabolites of MhuD, which have been proposed to meet the as cyclooxygenase (COX), is another type of dioxygenase where a physiological demands of the pathogenic bacterium. nonactivated O2 molecule binds to a fatty acid radical generated by initial hydrogen abstraction (6, 7). Whereas COX as well as Author contributions: T.M. and M.I.-S. designed research; T.M., S.N., S.T., and M.I.-S. per- linoleate diol synthase further convert the dioxygenated product formed research; C.W.G. and H.F. contributed new reagents/analytic tools; and T.M. and with peroxidase or isomerase activity, respectively (6, 8), each M.I.-S. wrote the paper. dioxygenase has another active site designed for the latter re- The authors declare no conflict of interest. action. Contrary to the conventional one active site, one reaction This article is a PNAS Direct Submission. 1To whom correspondence may be addressed. Email: [email protected] or paradigm, here we report that MhuD, a heme-degrading enzyme [email protected]. from Mycobacterium tuberculosis, catalyzes a sequential mono- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. oxygenation and dioxygenation in its single active site. 1073/pnas.1523333113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1523333113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 A OH + O O – O – 2e ,O2 O2 4e ,O2 N N N N N N e– NH N Fe3+ Fe3+ Fe3+ N N N N N N – NH HN H2O2 H2O2,2e CO HOOC COOH HOOC COOH HOOC COOH HOOC COOH Heme meso-Hydroxyheme Verdoheme Biliverdin BC OHC CHO O O Asn7 CN NH HN NH N O O N HN NH HN Heme Heme His75 HOOC COOH HOOC COOH Mycobilin-a Mycobilin-b Fig. 1. HO- and MhuD-type heme degrading enzymes. (A) Heme degradation mechanism of canonical HO enzymes with alternative H2O2 pathways. (B) Structures of mycobilin isomers. (C) Crystal structure of cyanide-bound ferric heme-MhuD [Protein Data Bank (PDB) ID code 4NL5]. Results Reduction of ferryl heme, which is also achieved in the presence First, we demonstrate that MhuD catalysis is initiated by re- of deferoxamine (Fig. S4B), allows the regenerated ferric heme to – ductive O2 activation (O2/e ), suggesting HO-type self-mono- cycle through another H2O2 reaction. If the H2O2 reaction af- oxygenation. Reduction of ferric heme-MhuD in the presence of fords a small amount of hydroxyheme via this pathway (Fig. S4E), O2 accumulates an intermediate with Soret and visible peaks at its accumulation should become detectable upon multiple turn- 407 and 560 nm, respectively (Fig. 2A). The same intermediate overs. In fact, a reaction of ferric heme-MhuD with a fourfold can be prepared by the reaction of ferrous heme-MhuD with O2 excess of H2O2 in the presence of deferoxamine results in the 18 (Fig. S2). Resonance Raman spectroscopy detects three O2- emergence of characteristic visible bands around 540 and 615 nm sensitive signals, suggesting that this metastable intermediate is (Fig. S4D), indicating partial accumulation of hydroxyheme. oxy-ferrous heme-MhuD (Fig. 2B and Table S1). Oxy-MhuD is The observed intermediate exhibits a similar reactivity as the further reduced to produce the mycobilin isomers in the pres- hydroxyheme complex of MhuD as described below. Thus, the ence of deferoxamine, a high-affinity iron chelator (Figs. 1B and initial step of MhuD heme degradation is the inefficient conven- 2C). We next find that H2O2 can substitute for the first O2 ac- tional self-monooxygenation of heme to hydroxyheme through – tivation (O2/e ) step, as expected for monooxygenation (19). The oxy-heme and possibly FeOOH-heme intermediates (Fig. 3) reaction of heme-MhuD and H2O2 in the absence of a reductant (20, 23, 24). results in mycobilin formation with a similar isomer ratio (Fig. The hydroxyheme oxidation leading to ring cleavage has been 2C and Fig. S3A). For H2O2-dependent mycobilin formation, reported to occur only at the hydroxylated site (on-site oxidation α there is a requirement for O2 (Fig. S3B), suggesting the transient in Fig. 3) (25, 26). Despite the -meso cleavage in both mycobilin formation of meso-hydroxyheme, which is similar to its conver- isomers (Fig. 1B), α-hydroxyheme-MhuD does not afford mycobilin sion in the HO second step that also strictly requires O2 (Fig. 1A) even in the presence of H2O2 and O2 (Fig. 2C and Fig. S5A). In- (19, 20). stead, β-hydroxyheme-MhuD produces mycobilin-a with the addi- β Formation of hydroxyheme from ferric heme-MhuD with H2O2 tional oxidation at the -meso carbon atom, whereas mycobilin-b was examined under anaerobic conditions. The primary product with the δ-meso oxidation is generated from δ-hydroxyheme-MhuD exhibited its Soret peak at 402 nm with a visible peak at 573 nm (Figs.
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