Toshitaka Matsui

Effects of the Location of Distal Histidine in the Reaction of Myoglobin with Hydrogen Peroxide

To clarify how the location of distal histidine affects the activation process of H2O2 by heme proteins, we have characterized reactions with H2O2 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 H2O2, 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 H2O2. 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 H2O2 by heme enzymes.

Heme Oxygenase Reveals Its Strategy for Catalyzing Three Successive Oxygenation Reactions

Heme oxygenase (HO) is an enzyme that catalyzes the regiospecific conversion of heme to biliverdin IXalpha, CO, and free iron. In mammals, HO has a variety of physiological functions, including heme catabolism, iron homeostasis, antioxidant defense, cellular signaling, and O(2) sensing. The enzyme is also found in plants (producing light-harvesting pigments) and in some pathogenic bacteria, where it acquires iron from the host heme. The HO-catalyzed heme conversion proceeds through three successive oxygenations, a process that has attracted considerable attention because of its reaction mechanism and physiological importance. The HO reaction is unique in that all three O(2) activations are affected by the substrate itself. The first step is the regiospecific self-hydroxylation of the porphyrin alpha-meso carbon atom. The resulting alpha-meso-hydroxyheme reacts in the second step with another O(2) to yield verdoheme and CO. The third O(2) activation, by verdoheme, cleaves its porphyrin macrocycle to release biliverdin and free ferrous iron. In this Account, we provide an overview of our current understanding of the structural and biochemical properties of the complex self-oxidation reactions in HO catalysis. The first meso-hydroxylation is of particular interest because of its distinct contrast with O(2) activation by cytochrome P450. Although most heme enzymes oxidize exogenous substrates by high-valent oxo intermediates, HO was proposed to utilize the Fe-OOH intermediate for the self-hydroxylation. We have succeeded in preparing and characterizing the Fe-OOH species of HO at low temperature, and an analysis of its reaction, together with mutational and crystallographic studies, reveals that protonation of Fe-OOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous auto-oxidation of the reactive alpha-meso-hydroxyheme; its mechanism remains elusive, but the HO enzyme has been shown not to play a critical role in it. Until recently, the means of the third O(2) activation had remained unclear as well, but we have recently untangled its mechanistic outline. Reaction analysis of the verdoheme-HO complex strongly suggests the Fe-OOH species as a key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, including the critical roles of the distal water molecule. A comprehensive study of the three oxygenations of HO highlights the rational design of the enzyme architecture and its catalytic mechanism. Elucidation of the last oxygenation step has enabled a kinetic analysis of the rate-determining step, making it possible to discuss the HO reaction mechanism in relation to its physiological functions.

Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, andSIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. EcDOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its α-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min−1 with cAMP, which was optimal at pH 8.5 in the presence of Mg2+ and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.

Structure and catalytic mechanism of heme oxygenase

Covering: up to 2006 Heme oxygenase (HO) catalyzes O2-dependent regiospecific conversion of heme to biliverdin, CO and free Fe(II). The heme group is tightly sandwiched between the “proximal” and “distal” helices with a neutral imidazole of His as an axial ligand. In the ferrous form, both helices move closer to the heme group, and O2 binds with an acute Fe–O–O angle of ∼110°, the distal helix restricts the O–O bond direction placing the terminal oxygen atom close to the α-meso-carbon. The bound O2 is stabilized by hydrogen bonds with a distal Gly amide nitrogen and the nearby H2O, the latter of which is a part of an extended distal pocket hydrogen bonding network linked by a conserved distal Asp. The hydrogen bonding network functions as a conduit for transferring protons required for the formation of the ferric hydroperoxo, generated by one-electron reduction of the oxy form, and also for the activation of the hydroperoxo, leading to the selective hydroxylation of the heme α-meso-carbon. The ferric hydroperoxo active species could not be formed upon loss of the nearby H2O, indicating a critical role of this H2O molecule in the meso-carbon hydroxylation. Ferrous verdoheme formation proceeds by reaction of the ferrous porphyrin neutral radical of ferric α-meso-hydroxyheme with O2 and one electron. Ferrous verdoheme iron reacts with O2 to form a reaction intermediate, reduction of which affords biliverdin. Proton transfer by the distal pocket hydrogen bonding network facilitates conversion of verdoheme to biliverdin. HO heme catabolism is realized by the salient HO protein structure that enables conversion of heme, which is rather inert, into reactive hydroxyheme and verdoheme intermediates.

Rational molecular design of a catalytic site: engineering of catalytic functions to the myoglobin active site framework

Abstract Proteins that contain the heme prosthetic group are responsible for many different types of catalytic activity. Understanding the mechanisms through which a particular type of catalytic activity is favored over the others remains a significant challenge. Recently, the most common strategy for structure–function studies for a particular enzyme has involved substitution of amino acid residues by site-directed mutagenesis followed by investigations of the effect of the substitution on the catalytic activity of that system. This work describes a significant departure from this common strategy. Instead, we seek to convert a non-enzymatic hemoprotein into one that is capable of catalytic activity. In so doing, we expect to gain an understanding of the general structural requirements for particular enzymatic functions. Comparison of X-ray crystal structures of myoglobin and peroxidases reveals differences in arrangement of amino acid residues in the heme pockets. On the basis of these structural differences and the reaction mechanism of peroxidases, we have rationally designed several myoglobin mutants in order to convert myoglobin into a peroxidase-like enzyme. We have discovered that the location of the distal histidine in the active site provides a critical balance between the formation and subsequent decay of the oxo-ferryl porphyrin radical cation (compound I), a catalytic species for one- and two-electron oxidation and oxygen transfer reactions. The mutants prepared in this work have been altered in such a manner that they have permitted compound I to be observed in myoglobin for the first time. This allows us to investigate mechanistic details under single turnover conditions by use of double mixing stopped-flow spectroscopy. Furthermore, some of the mutants we have constructed might be useful as good catalysts for asymmetric oxidations. In this short review, we describe our attempts to elucidate structure–function relationships on the activation of the oxygen–oxygen bond of peroxides by hemoproteins.

Bach1, a heme‐dependent transcription factor, reveals presence of multiple heme binding sites with distinct coordination structure

The mammalian transcription factor Bach1 functions as a repressor of the enhancers of heme oxygenase‐1 (HO‐1) gene (Hmox‐1) by forming heterodimers with the small Maf proteins such as MafK. The transcription of Hmox‐1 is regulated by the substrate of HO‐1, heme. Heme induces expression of Hmox‐1 in part by inhibiting the binding of Bach1 to the enhancers and inducing the nuclear export of Bach1. A dipeptide motif of cysteine and proline (CP motif) in Bach1 is essential for the heme‐mediated regulation. In this study, we show that five molecules of heme bind to Bach1 by the heme‐titration assay. The Bach1‐heme complex exhibits an absorption spectrum with a major Soret peak at 371 nm and Raman band at 343 cm‐1 in high amounts of heme and a spectrum containing the major Soret peak at 423 nm at low heme concentrations. The spectroscopic characterization indicates that Bach1 has two kinds of heme‐binding sites with different coordination structures. Mutagenesis studies have established that four molecules of heme bind to the cysteine residues of four CP motifs in the C terminus of Bach1. These results raise the possibility that two separated activities of Bach1, DNA‐binding and nuclear export, are regulated by heme binding at the different CP motifs of Bach1 respectively, but not by cooperative heme‐binding.

A new way to degrade heme: the Mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO.

Background: IsdG is a novel heme-degrading enzyme found in pathogenic bacteria. Results: MhuD, an IsdG-type enzyme from Mycobacterium tuberculosis, degrades heme into unusual tetrapyrroles without generating carbon monoxide. Conclusion: The unique MhuD reaction is mechanistically distinct from that of canonical heme oxygenase enzymes. Significance: Nonplanarity of heme in the IsdG-type enzymes appears to cause a new degradation pathway. MhuD is an oxygen-dependent heme-degrading enzyme from Mycobacterium tuberculosis with high sequence similarity (∼45%) to Staphylococcus aureus IsdG and IsdI. Spectroscopic and mutagenesis studies indicate that the catalytically active 1:1 heme-MhuD complex has an active site structure similar to those of IsdG and IsdI, including the nonplanarity (ruffling) of the heme group bound to the enzyme. Distinct from the canonical heme degradation, we have found that the MhuD catalysis does not generate CO. Product analyses by electrospray ionization-MS and NMR show that MhuD cleaves heme at the α-meso position but retains the meso-carbon atom at the cleavage site, which is removed by canonical heme oxygenases. The novel tetrapyrrole product of MhuD, termed “mycobilin,” has an aldehyde group at the cleavage site and a carbonyl group at either the β-meso or the δ-meso position. Consequently, MhuD catalysis does not involve verdoheme, the key intermediate of ring cleavage by canonical heme oxygenase enzymes. Ruffled heme is apparently responsible for the heme degradation mechanism unique to MhuD. In addition, MhuD heme degradation without CO liberation is biologically significant as one of the signals of M. tuberculosis transition to dormancy is mediated by the production of host CO.

Dioxygen Activation for the Self-Degradation of Heme: Reaction Mechanism and Regulation of Heme Oxygenase

Heme oxygenase (HO) catalyzes the regiospecific conversion of heme to biliverdin, CO, and free iron through three successive oxygenation reactions. HO catalysis is unique in that all three O(2) activations are performed by the substrate itself. This Forum Article overviews our current understanding on the structural and biochemical properties of HO catalysis, especially its first and third oxygenation steps. The HO first step, regiospecific hydroxylation of the porphyrin alpha-meso-carbon atom, is of particular interest because of its sharp contrast to O(2) activation by cytochrome P450. HO was proposed to utilize the FeOOH species but not conventional ferryl hemes as a reactive intermediate for self-hydroxylation. We have succeeded in preparing and characterizing the FeOOH species of HO at low temperature, and our analyses of its reaction, together with mutational and crystallographic studies, reveal that protonation of FeOOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous autooxidation of the reactive alpha-meso-hydroxyheme in which the HO enzyme does not play a critical role. Further O(2) activation by verdoheme cleaves its porphyrin macrocycle to form biliverdin and free ferrous iron. This third step has been considered to be a major rate-determining step of HO catalysis to regulate the enzyme activity. Our reaction analysis strongly supports the FeOOH verdoheme as the key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, and the distal water is suggested to enhance the third step as expected from the similarity. The HO mechanistic studies highlight the catalytic importance of the distal hydrogen-bonding network, and this manuscript also involves our attempts to develop HO inhibitors targeting the unique distal structure.

Heme degradation by Staphylococcus aureus IsdG and IsdI liberates formaldehyde rather than carbon monoxide.

IsdG and IsdI from Staphylococcus aureus are novel heme-degrading enzymes containing unusually nonplanar (ruffled) heme. While canonical heme-degrading enzymes, heme oxygenases, catalyze heme degradation coupled with the release of CO, in this study we demonstrate that the primary C1 product of the S. aureus enzymes is formaldehyde. This finding clearly reveals that both IsdG and IsdI degrade heme by an unusual mechanism distinct from the well-characterized heme oxygenase mechanism as recently proposed for MhuD from Mycobacterium tuberculosis. We conclude that heme ruffling is critical for the drastic mechanistic change for these novel bacterial enzymes.

Asymmetric oxidation catalyzed by myoglobin mutants

Abstract The sperm whale myoglobin active site mutants (L29H/H64L and F43H/H64L Mb) have been shown to catalyze the asymmetric oxidation of sulfides and olefins. Thioanisole, ethyl phenyl sulfide, and cis -β-methylstyrene are oxidized by L29H/H64L Mb with more than 95% enantiomeric excess (% ee). On the other hand, the F43H/H64L mutant transforms trans -β-methylstyrene into the trans -epoxide with 96% ee. The dominant sulfoxide product in the incubation of alkyl phenyl thioethers is the R isomer; however, the mutants afford dominantly the S isomer of aromatic bicyclic sulfoxides. The results help us to rationalize the difference in the preferred stereochemistry of the Mb mutant-catalyzed reactions. Furthermore, the Mb mutants exhibit an improvement in the oxidation rate up to 300-fold with respect to wild type.