The relationship between liquid-liquid phase separation and microheterogeneity in water-primary alcohol mixtures was examined by analysing the mass spectra of clusters generated through the fragmentation of liquid droplets. By comparing the cluster structures of water-ethanol, -1-propanol, and -1-butanol binary mixtures at various alcohol concentrations, we discovered differences in the molecular clusters that control phase separation. We also studied the role of water in alcohol self-association. Alcohol self-association is promoted in the presence of a small amount of water (ca. 10 approximately 20 wt%), in which the water-water hydrogen-bonding network is weak and does not contribute to alcohol self-association. We have demonstrated that alcohol self-association is also promoted by non-ideal mixing with other alcohols. The self-association of alcohol molecules complements the loss of stabilization energy caused by the relatively weak coexisting interactions. This complementary relationship among intermolecular interactions is an inherent property of solutions, and plays a key role in the phase separation process.

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Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

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 ...

Aspects of protein engineering of cytochrome P450 (P450) and myoglobin (Mb) to construct selective oxygenation catalysts have been described. Heme enzymes are known as biocatalysts for various oxidations but the design of substrate specificity has still remained one of the significant challenges because of dynamic nature of enzyme-substrate interactions. In particular, P450s are the most interesting targets among the heme enzymes because they are able to catalyze many types of monooxygenations such as hydroxylation, epoxidation, and sulfoxidation with high selectivity. Thus, many researchers have made efforts to convert the selectivity for natural substrates into that for unnatural substrates by several protein engineering approaches. On the other hand, we have reported a rational design of Mb to convert its oxygen carrier function into that of peroxidase or peroxygenase. The Mb mutants prepared in our work afford oxo-ferryl porphyrin radical cation (compound I) as observable species in Mb for the first time. Furthermore, some of the mutants we have constructed are useful for enantioselective oxygenations by oxygen transfer from the Mb-compound I to substrates.