Aerobic life is possible because the molecular structure of oxygen (O2) makes direct reaction with most organic materials at ambient temperatures an exceptionally slow process. Of course, these reactions are inherently very favorable, and they occur rapidly with the release of a great deal of energy at high temperature. Nature has been able to tap this sequestered reservoir of energy with great spatial and temporal selectivity at ambient temperatures through the evolution of oxidase and oxygenase enzymes. One mechanism used by these enzymes for O2 activation has been studied in detail for the soluble form of the enzyme methane monooxygenase. These studies have revealed the step-by-step process of O2 activation and insertion into the ultimately stable C–H bond of methane. Additionally, an elegant regulatory mechanism has been defined that enlists size selection and quantum tunneling to allow methane oxidation to occur specifically in the presence of more easily oxidized substrates.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high-resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O2 access routes enhanced when the complex forms. Previously reported, lower-resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O2 conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O2 conduits in the M. trichosporium OB3b sMMOH:MMOB complex revealed a narrow molecular tunnel, termed the W308-tunnel. This tunnel is sized appropriately for O2 and traverses the sMMOH–MMOB interface before accessing the active site. The kinetics of reaction of O2 with the diferrous sMMOH:MMOB complex in solution show that use of the MMOB V41R variant decreases the rate constant for O2 binding >25000-fold without altering the component affinity. The location of Val41 near the entrance to the W308-tunnel is consistent with the tunnel serving as the primary route for the transfer of O2 into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-tunnel.

Infrared spectroscopic ellipsometry was used for determination of molecular orientation and for lateral homogeneity studies of organic monolayers on GaAs and Au, the organic layer being either octanedithiol or hexadecanethiol (HDT). The laterally resolved measurements were performed with the infrared mapping ellipsometer at the synchrotron storage ring BESSY II. The molecular orientation within the monolayers was determined by optical model simulations of the measured ellipsometric spectra. Different tilt angles were obtained for the monolayers of HDT and octanedithiol on GaAs: 19 degrees and >30 degrees , respectively. The tilt angle of the methylene chains for HDT on Au substrate (22 degrees ) is similar to the 19 degrees tilt which was obtained for the HDT monolayers on GaAs, thus suggesting similar molecular ordering of the thiolates on both substrates.

Clinical studies to assess the benefits of blood transfusion or haemodilution in critical illness should take account of measured CBV before, during and after intervention. As mentioned above, surrogate measures of CBV are inadequate and studies based on these must be considered incomplete, because they cannot distinguish between effects of changes in haemoglobin concentration and changes in blood volume. The choice of a suitable technique for measuring CBV depends on the facilities available locally. In general, methods based on labelled red cells are more reliable but are technically demanding and time consuming. Those based on albumin are likely to yield false high values and this is particularly true in all patients with impaired capillary integrity. The most promising plasma marker is hydroxyethyl starch which may be particularly useful when the polysaccharide is labelled with a fluorescent dye. Attaching fluorescein to hydroxyethyl starch is not difficult and, should demand be sufficient, it may well become available from manufacturers who are already capable of providing other fluorescent polysaccharides. The clinical benefits of such a development would include more rational schedules of i.v. fluid and blood transfusion management in surgical and intensive care patients.

Nanoparticles resistant to salt-induced aggregation are continually being developed for biomedical and industrial applications. Because of their colloidal stability these functionalized nanoparticles are anticipated to be persistent aquatic contaminants. Here, we show that Corbicula fluminea, a globally distributed clam that is a known sentinel of aquatic ecosystem contamination, can uptake and biodeposit bovine serum albumin (BSA) stabilized gold nanoparticles. Nanoparticle clearance rates from suspension were dictated by diameter and concentration, with the largest particles cleared most quickly on a mass basis. Particle capture facilitates size-selective 'biopurification' of particle suspensions with nanoscale resolution. Nanoparticles were retained either within the clam digestive tract or excreted in feces. Our results suggest that biotransformation and biodeposition will play a significant role in the fate and transport of persistent nanoparticles in aquatic systems.

We have investigated the reaction pathways involved in the electron-beam-induced damage of −CH3, −OH, and −COOH terminated alkanethiolate self-assembled monolayers (SAMs) adsorbed on GaAs (001) using time-of-flight secondary ion mass spectrometry. Upon electron beam exposure, the monolayers dehydrogenate, leading to the formation of CC bonds, cross-links, and polycyclic aromatic hydrocarbons (PAHs). We also observe C−S bond scission. The data suggest that the electron beam damage is not uniform along the alkanethiol backbone. Upon electron beam exposure, we observe the loss of CO2 and H2O from the −COOH and −OH terminated SAMs, respectively, indicating that the terminal groups decompose. Although the SAM degradation mechanism is similar to that previously reported for alkanethiolate SAMs adsorbed on metals, there are some important differences. First, the electron dose required to form CC bonds and PAHs is much smaller than for SAMs adsorbed on metals. Second, although we observe C−S bond scission, we do not observe As−S or Ga−S bond scission and there is no desorption of S, or S-containing, fragments from the surface. We discuss the implications of these studies on the optimization of the experimental conditions for electron beam lithography using SAM resists on semiconductors.

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