Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.

The heterolytic and homolytic N-NO bond dissociation energies of seven substituted N-methyl-N-nitrosobenzenesulfonamides (abbreviated as G-MNBS, G = p-OCH(3), p-CH(3), p-H, p-Cl, p-Br, 2,5-2Cl, m-NO(2)) in acetonitrile solution were evaluated for the first time by using titration calorimetry and relative thermodynamic cycles according to Hess' law. The results show that the energetic scales of the heterolytic and homolytic N-NO bond dissociation energies of G-MNBS in acetonitrile solution cover the ranges from 44.3 to 49.5 and from 33.0 to 34.9 kcal/mol for the neutral G-MNBS, respectively, which indicates that N-methyl-N-nitrosobenzenesulfonamides are much easier to release a NO radical (NO(*)) than to release a NO cation (NO(+)). The estimation of the heterolytic and homolytic (N-NO)(-)(*) bond dissociation energies of the seven G-MNBS radical anions in acetonitrile solution gives the energetic ranges of -15.8 to -12.9 and -3.1 to 1.8 kcal/mol for the (N-NO)(-)(*) bond homolysis and heterolysis, respectively, which means that G-MNBS radical anions are very unstable at room temperature and able to spontaneously or easily release a NO radical or NO anion (NO(-)), but releasing a NO radical is easier than releasing NO anion. These determined N-NO bond dissociation energies of G-MNBS and their radical anions have been successfully used in the mechanism analyses of NO transfer from G-MNBS to 3,6-dibromocarbazole and the reactions of NO with the substituted N-methyl-benzenesulfonamide nitranions (G-MBSN(-)) in acetonitrile solution.

Addition of a second coordinating agent (coord*) to a solution of RM(coord) + A -(R ) ethyl or neopentyl, M ) Zn or Mg, A -) 1,2,3,4-tetraphenylcyclopentadienyl) can provide equilibrium mixtures of these compounds, coord, and RM(coord*) + A -. This exchange with RMg(coord) + requires the addition of a small amount of R 2 Mg, but added R 2 Zn is not necessary for exchanges with RZn(coord) + . The equilibrium constants provide information about the relative abilities of different coordinating agents to coordinate to RM + and reveal significant differences between coordination to RMg + and RZn + . Reactions of RM(coord) + with R′ 2 M (R ) ethyl or neopentyl for RMg(coord) + and ethyl, isopropyl, tert-butyl, neopentyl, or p-methylphenyl for RZn(coord) + ) provide equilibrium mixtures of these components, R′M-(coord) + , and R 2 M. The equilibrium constants provide information about the effect of R on stability. An X-ray structure of p-methylphenylzinc (2,5,8,11-tetramethyl-2,5,8,11-tetraazadodecane) + shows that just three of the N atoms are coordinated to Zn. The effects of coord, R, and metal on RM(coord) + stability are discussed, and the abilities of coordinating agents to coordinate to RM + , to slow allylic isomerization of (CH 2 dCMeCH 2 ) 2 Zn, and to convert R 2 Zn to RZn(coord) + are compared.Coordination plays a major role in organomagnesium and organozinc chemistry. Because of a strong propensity to form additional bonds, the metal atoms of the compounds, usually written as RMgX, R 2 Mg, RZnX, and R 2 Zn (X a halogen, R alkyl or aryl), generally coordinate to functions such as alkoxy or amino when these are present, often as part of the solvent. 3,4 Additional bonds also may result from bridging of groups between metal atoms. Formation of several strong bonds between O or N atoms of a macrocycle and the Mg of the cation must provide the driving force for reactions of some macrocycles with organomagnesium compounds to produce coordinated RMg + cations and organomagnesate anions, as exemplified in eq 1. [5][6][7][8] Because of such observations, we wanted to determine the abilities of coordinating

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