Seho Lee

Three new intermediates of the catalytic cycle of the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been detected using transient kinetic techniques. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains an oxygen-bridged [Fe(III).Fe(III)] cluster that catalyzes O2 activation and insertion chemistry. At 4 degrees C, rapid mixing of O2 with diferrous MMOH ([Fe(II).Fe(II)]) in the presence of a 2-fold excess of MMOB resulted in loss of the g = 16 EPR signal characteristic of the diferrous cluster at an apparent first order rate of 22 +/- 5 s-1 (O2 approximately 700 microM). Subsequently, an EPR silent, chromophoric (lambda max = 330 and 430 nm, epsilon approximately 7500 M-1 cm-1 at each wavelength) intermediate (compound Q) formed with an average first order rate constant of 1 +/- 0.1 s-1 and then decayed at 0.05 +/- 0.01 s-1. Since compound Q formed much more slowly than diferrous MMOH disappeared, at least one other undetected intermediate (compound P) must have formed before compound Q. MMO substrates had little or no effect on the formation rate of compound Q, but they caused the decay rate to increase linearly with the concentration added. The substrates methane, furan, and nitrobenzene caused compound Q decay to occur with second order rate constants of 19,000 M-1 s-1, 9000 M-1 s-1, and 200 M-1 s-1 (+/- 5%), respectively. When nitrobenzene was used as a substrate, a second chromophoric intermediate (compound T, lambda max = 325 nm, with a shoulder at 395 nm, epsilon 395 approximately 6000 M-1 cm-1) formed at the same rate as compound Q decay. Chemical quench studies showed that compound T is an enzyme-product complex that decays with a rate constant of 0.02 +/- 0.005 s-1. This rate is approximately the same as kcat for nitrobenzene turnover at 4 degrees C catalyzed by the reconstituted MMO system, suggesting that product release is the rate-limiting step in catalysis. The characteristics of compound Q suggest that it may be the activated form of the enzyme that directly catalyzes substrate oxidation.

The soluble methane monooxygenase (MMO) system, consisting of reductase, component B, and hydroxylase (MMOH), catalyzes NADH and O2-dependent monooxygenation of many hydrocarbons. MMOH contains 2 mu-(H or R)oxo-bridged dinuclear iron clusters thought to be the sites of catalysis. Although rapid NADH-coupled turnover requires all three protein components, three less complex systems are also functional: System I, NADH, O2, reductase, and MMOH; System II, H2O2 and oxidized MMOH; System III, MMOH reduced nonenzymatically by 2e- and then exposed to O2 (single turnover). All three systems give the same products, suggesting a common reactive oxygen species. However, the distribution of products observed for most substrates that are hydroxylated in more than one position is different for each system. For several of these substrates, addition of component B to Systems I, II, or III causes the product distributions to shift dramatically. These shifts result in identical product distributions for Systems I and III in which MMOH passes through the 2e- reduced state ([Fe(II).Fe(II)]) during catalysis. In contrast, System II (in which MMOH probably does not become reduced) generally gives a unique product distribution. It is proposed that changes in MMOH structure occurring upon diiron cluster reduction and/or component complex formation cause substrates to be presented differently to the activated oxygen species. Kinetic studies show that component B strongly activates System I and, in most cases, strongly deactivates System II. The effect of component B on product distribution of System I (and III) occurs at less than 5% of the MMOH concentration, while nearly stoichiometric concentrations are required to maximize the rate of System I. This shows that component B has at least two roles in catalysis. EPR monitored titration of reduced MMOH ([Fe(II).Fe(II)]) with component B suggests that the effect of substoichiometric component B on product distribution is due to hysteresis in the MMOH conformational changes.

A self‐ordering system based on anti‐streptavidin viruses is presented. A variety of materials, including inorganic nanoparticles, small organic molecules, and large biomolecules, can be organized at the nanometer length scale using this method. Molecular recognition is used to bind nanosize materials (e.g., gold nanoparticles) to the virus (see cover) and the resulting complex spontaneously evolves into a self‐supporting hybrid film (see Figure).

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5.2. Enzyme activity was optimal at 50 degrees C, and at pH 5.5. The enzyme was stable at 20-40 degrees C, and at pH 5-6 with a K(m) value of 2.4 g l-1 soluble starch. The activation energy was 42.3 kJ mol-1. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu2+ (5 mmol l-1), Zn2+ (5 mmol l-1), N-bromosuccinimide (5 mmol l-1), EDTA (5 mmol l-1), I2 (1 mmol l-1) and activated by beta-mercaptoethanol (10 mmol l-1).