8 publications

8 publications

A Hydrogenase Model System Based on the Sequence of Cytochrome c: Photochemical Hydrogen Evolution in Aqueous Media

Hayashi, T

Chem. Commun. 2011, 47, 8229, 10.1039/c1cc11157d

The diiron carbonyl cluster is held by a native CXXC motif, which includes Cys14 and Cys17, in the cytochrome c sequence. It is found that the diiron carbonyl complex works well as a catalyst for H2 evolution. It has a TON of ∼80 over 2 h at pH 4.7 in the presence of a Ru-photosensitizer and ascorbate as a sacrificial reagent in aqueous media.


Metal: Fe
Ligand type: Carbonyl
Host protein: Cytochrome c
Anchoring strategy: Dative
Optimization: ---
Reaction: H2 evolution
Max TON: 82
ee: ---
PDB: ---
Notes: Horse heart cytochrome C

A Rhodium Complex-Linked β-Barrel Protein as a Hybrid Biocatalyst for Phenylacetylene Polymerization

Hayashi, T

Chem. Commun. 2012, 48, 9756, 10.1039/C2CC35165J

Our group recently prepared a hybrid catalyst containing a rhodium complex, Rh(Cp)(cod), with a maleimide moiety at the peripheral position of the Cp ligand. This compound was then inserted into a β-barrel protein scaffold of a mutant of aponitrobindin (Q96C) via a covalent linkage. The hybrid protein is found to act as a polymerization catalyst and preferentially yields trans-poly(phenylacetylene) (PPA), although the rhodium complex without the protein scaffold normally produces cis PPA.


Metal: Rh
Ligand type: COD; Cp*
Host protein: Nitrobindin (Nb)
Anchoring strategy: Cystein-maleimide
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Crystal Structure and Peroxidase Activity of Myoglobin Reconstituted with Iron Porphycene

Hayashi, T

Inorg. Chem. 2006, 45, 10530-10536, 10.1021/ic061130x

The incorporation of an artificially created metal complex into an apomyoglobin is one of the attractive methods in a series of hemoprotein modifications. Single crystals of sperm whale myoglobin reconstituted with 13,16-dicarboxyethyl-2,7-diethyl-3,6,12,17-tetramethylporphycenatoiron(III) were obtained in the imidazole buffer, and the 3D structure with a 2.25-Å resolution indicates that the iron porphycene, a structural isomer of hemin, is located in the normal position of the heme pocket. Furthermore, it was found that the reconstituted myoglobin catalyzed the H2O2-dependent oxidations of substrates such as guaiacol, thioanisole, and styrene. At pH 7.0 and 20 °C, the initial rate of the guaiacol oxidation is 11-fold faster than that observed for the native myoglobin. Moreover, the stopped-flow analysis of the reaction of the reconstituted protein with H2O2 suggested the formation of two reaction intermediates, compounds II- and III-like species, in the absence of a substrate. It is a rare example that compound III is formed via compound II in myoglobin chemistry. The enhancement of the peroxidase activity and the formation of the stable compound III in myoglobin with iron porphycene mainly arise from the strong coordination of the Fe−His93 bond.


Metal: Fe
Ligand type: Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: 1MBI
Notes: ---

C(sp3)–H Bond Hydroxylation Catalyzed by Myoglobin Reconstituted with Manganese Porphycene

Hayashi, T

J. Am. Chem. Soc. 2013, 135, 17282-17285, 10.1021/ja409404k

Myoglobin reconstituted with manganese porphycene was prepared in an effort to generate a new biocatalyst and was characterized by spectroscopic techniques. The X-ray crystal structure of the reconstituted protein reveals that the artificial cofactor is located in the intrinsic heme-binding site with weak ligation by His93. Interestingly, the reconstituted protein catalyzes the H2O2-dependent hydroxylation of ethylbenzene to yield 1-phenylethanol as a single product with a turnover number of 13 at 25 °C and pH 8.5. Native myoglobin and other modified myoglobins do not catalyze C–H hydroxylation of alkanes. Isotope effect experiments yield KIE values of 2.4 and 6.1 for ethylbenzene and toluene, respectively. Kinetic data, log kobs versus BDE(C(sp3)–H) for ethylbenzene, toluene, and cyclohexane, indicate a linear relationship with a negative slope. These findings clearly indicate that the reaction occurs via a rate-determining step that involves hydrogen-atom abstraction by a Mn(O) species and a subsequent rebound hydroxylation process which is similar to the reaction mechanism of cytochrome P450.


Metal: Mn
Ligand type: Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Reaction: Hydroxylation
Max TON: ---
ee: ---
PDB: 2WI8
Notes: ---

Generation of New Artificial Metalloproteins by Cofactor Modification of Native Hemoproteins

Review

Hayashi, T

Isr. J. Chem. 2015, 55, 76-84, 10.1002/ijch.201400123

Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O2 storage protein, can be reconstituted with iron porphycene to dramatically enhance the O2 affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H2O2‐dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert CH bond. It is also of interest to insert a non‐porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β‐barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.


Notes: ---

Meso-Unsubstituted Iron Corrole in Hemoproteins: Remarkable Differences in Effects on Peroxidase Activities between Myoglobin and Horseradish Peroxidase

Hayashi, T

J. Am. Chem. Soc. 2009, 131, 15124-15125, 10.1021/ja907428e

Myoglobin (Mb) and horseradish peroxidase (HRP) were both reconstituted with a meso-unsubstituted iron corrole and their electronic configurations and peroxidase activities were investigated. The appearance of the 540 nm band upon incorporation of the iron corrole into apoMb indicates axial coordination by the proximal histidine imidazole in the Mb heme pocket. Based on 1H NMR measurements using the Evans method, the total magnetic susceptibility of the iron corrole reconstituted Mb was evaluated to be S = 3/2. In contrast, although a band does not appear in the vicinity of 540 nm during reconstitution of the iron corrole into the matrix of HRP, a spectrum similar to that of the iron corrole reconstituted Mb is observed upon the addition of dithionite. This observation suggests that the oxidation state of the corrole iron in the reconstituted HRP can be assigned as +4. The catalytic activities of both proteins toward guaiacol oxidation are quite different; the iron corrole reconstituted HRP decelerates H2O2-dependent oxidation of guaiacol, while the same reaction catalyzed by iron corrole reconstituted Mb has the opposite effect and accelerates the reaction. This finding can be attributed to the difference in the oxidation states of the corrole iron when these proteins are in the resting state.


Metal: Fe
Ligand type: Corrole
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Metal: Fe
Ligand type: Corrole
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Porphyrinoid Chemistry in Hemoprotein Matrix:  Detection and Reactivities of Iron(IV)-Oxo Species of Porphycene Incorporated into Horseradish Peroxidase

Hayashi, T

J. Am. Chem. Soc. 2007, 129, 12906-12907, 10.1021/ja074685f

The iron porphycene with two propionates at the peripheral positions of the framework was incorporated into the heme pocket of horseradish peroxidase. In the presence of hydrogen peroxide, the ferric iron porphycene was smoothly converted into the iron(IV)-oxo porphycene π-cation radical species, which was confirmed by the appearance of a band around 800 nm in the UV−vis spectrum. The protein with the iron porphycene showed a 10-fold higher reactivity for the thioanisole oxidation when compared to the native protein. In contrast, the guaiacol oxidation proceeded with similar reaction rates in both proteins. The kinetic analyses indicated that the ferric porphycene in the protein more slowly reacts with hydrogen peroxide than the native heme, whereas the high oxidation states show higher reactivities during oxidations of an organic substrate. The formation of the iron(IV)-oxo species of porphycene and its reactivities in the hemoprotein matrix are demonstrated.


Metal: Fe
Ligand type: Porphycene
Anchoring strategy: Reconstitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids

Review

Hayashi, T

Methods Enzymol. 2016, 439-454, 10.1016/bs.mie.2016.05.049

An important strategy used in engineering of hemoproteins to generate artificial enzymes involves replacement of heme with an artificial cofactor after removal of the native heme cofactor under acidic conditions. Replacement of heme in an enzyme with a nonnatural metalloporphyrinoid can significantly alter the reactivity of the enzyme. This chapter describes the design and synthesis of three types of artificial metalloporphyrinoid cofactors consisting of mono-, di-, and tri-anionic ligands (tetradehydrocorrin, porphycene, and corrole, respectively). In addition, practical procedures for the preparation of apo-hemoproteins, incorporation of artificial cofactors, and characterization techniques are presented. Furthermore, the representative catalytic activities of artificial enzymes generated by reconstitution of hemoproteins are summarized.


Notes: Book chapter