Experimental SectionMaterials. The pUC57 plasmid containing optimized nitrobindin (NB) (M75L, M148L) gene was purchased from GeneScript. Oligonucleotides were obtained from Invitrogen, Inc. (Japan). Restriction enzymes are obtained from Takara Bio Inc. (Japan). Nucleotide sequences were determined by Fasmac Co., Ltd (Japan). All reagents of the highest guaranteed grade were purchased and used as received unless otherwise noted. A standard rhodium solution for inductively coupled plasma optical emission spectroscopy (ICP-OES) was purchased from Wako (Japan). Distilled water was demineralized using a Barnstead NANOpure DIamond TM apparatus. 3-carboxy-1,3-propanedithiol S1 and {-(SCH 2 ) 2 CHCO 2 H}[Fe 2 (CO) 6 ] S2 were synthesized as described in a previous report.Instruments. 1 H and 13 C NMR spectra were recorded on a Bruker DPX400 NMR spectrometer. Chemical shifts were reported in ppm relative to the residual solvent resonances. ESI-TOF MS

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 H(2) 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.

A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis­(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.

In nature, heme cofactor-containing proteins participate not only in electron transfer and O 2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H 2 O 2 and O 2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal−oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp 3 )−H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a β-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C−C bond formation. The hydrophobic β-barrel structure plays a significant role in substrate binding as well as controlling the stereoselec...

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.

13C CP/MAS NMR and FE/TEM measurements of the aragonite brick of the nacreous layer of Pinctada fucata indicate that it assembles with highly oriented aragonite nanocrystals, which are regulated by biopolymers.

Alternating: a cofactor dyad consisting of a heme group (red in picture) and a bis(biotin) unit (blue) was synthesized and shown to specifically bind to both apomyoglobin and streptavidin. In the presence of the dyad, the 1:1 association of a disulfide-bridged myoglobin dimer (green) with streptavidin (gray) afforded a submicrometer-sized fibrous alternating copolymer.

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