Hydrated ionic liquids (ILs) were prepared by adding appropriate amounts of water to hydrophilic ILs. Some hydrated ILs show excellent solubilizing ability for proteins, keeping the basic properties of ILs. The solubility of cytochrome c (cyt c) depended on the structure of the component ions. When component anions have oxo acid residues, the resulting hydrated ILs solubilize cyt c quite well. In such hydrated ILs, the structure and activity of cyt c is influenced by the kosmotropicity of the component ions. We synthesized ILs from various ions having different kosmotropicity, including dihydrogen phosphate (dhp), dibutylphosphate, acetate, lactate, and methanesulfonate as anions. The activity of the dissolved cyt c depends on the permutations of kosmotropicity of the component ions. cyt c shows no structural change and retains its activity when dissolved in the hydrated choline dhp, which is an excellent combination of chaotropic cation and kosmotropic anion. Furthermore, cyt c dissolved in the hydrated choline dhp remained in a native state and was active after 18 months of storage at room temperature.

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (g av ‫؍‬ 2.0055) characteristic of phenoxyl -radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E 1/2 ‫؍‬ 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.The white-rot wood-metabolizing basidiomycete fungi are major degraders of lignin contributing essential chemistry to the global carbon cycle. Phanerochaete chrysosporium, the organism most extensively studied for its lignin-degrading ability, produces three classes of extracellular enzyme under ligninolytic conditions: lignin peroxidase, manganese peroxidase, and glyoxal oxidase (1, 2). In the presence of H 2 O 2 (3), lignin peroxidases oxidize and partially depolymerize lignin or lignin model compounds (4 -9). The oxidizing peroxide cosubstrate for this reaction must be generated in situ for efficient turnover of extracellular lignin peroxidase, a function performed by glyoxal oxidase, which catalyzes the oxidation of a number of aldehyde and ␣-hydroxy carbonyl compounds, reducing O 2 to H 2 O 2 in the process. The enzyme exhibits a broad substrate specificity for oxidation of simple aldehydes to the corresponding carboxylic acids, as shown by Reaction 1 (10).Two of the substrates for glyoxal oxidase (glyoxal (OHCCHO) and methylglyoxal (CH 3 COCHO)) are found in extracellular fluid of ligninolytic cultures (10) and are likely to represent physiological substrates for the enzyme in a complex extracellular metabolic scheme (9, 10). Purified glyoxal oxidase is catalytically inactive but can be activated by lignin peroxidase (11, 12), suggesting a possible extracellular regulatory circuit for the control of H 2 O 2 production by glyoxal oxidase, and lignin peroxidase activity by H 2 O 2 .P. chrysosporium glyoxal oxidase has been purified to homogeneity (11), cloned for high level expression in Aspergillus nidulans (13...

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