Myoglobin (Mb) is perhaps the most studied protein, experimentally and theoretically. Despite the wealth of known details regarding the gas migration processes inside Mb, there exists no fully conclusive picture of these pathways. We address this deficiency by presenting a complete map of all the gas migration pathways inside Mb for small gas ligands (O2, NO, CO, and Xe). To accomplish this, we introduce a computational approach for studying gas migration, which we call implicit ligand sampling. Rather than simulating actual gas migration events, we infer the location of gas migration pathways based on a free-energy perturbation approach applied to simulations of Mb's dynamical fluctuations at equilibrium in the absence of ligand. The method provides complete three-dimensional maps of the potential of mean force of gas ligand placement anywhere inside a protein-solvent system. From such free-energy maps we identify each gas docking site, the pathways between these sites, to the heme and to the external solution. Our maps match previously known features of these pathways in Mb, but also point to the existence of additional exits from the protein matrix in regions that are not easily probed by experiment. We also compare the pathway maps of Mb for different gas ligands and for different animal species.

Depression is a debilitating mental illness with clear developmental patterns from childhood through late adolescence. Here, we present data from the Gene Environment Mood (GEM) study, which used an accelerated longitudinal cohort design with youth (N = 665) starting in 3rd, 6th, and 9th grades, and a caretaker, who were recruited from the general community, and were then assessed repeatedly via semi-structured diagnostic interviews every 6-months over 3 years (7 waves of data) to establish and then predict trajectories of depression from age 8 to 18. First, we demonstrated that overall prevalence rates of depression over time, by age, gender, and pubertal status, in the GEM study closely match those trajectories previously obtained in past developmental epidemiological research. Second, we tested whether a genetic vulnerability-stress model involving 5-HTTLPR and chronic peer stress was moderated by developmental factors. Results showed that older aged adolescents with SS/SL genotype, who experienced higher peer chronic stress over 3 years, were the most likely to be diagnosed with a depressive episode over time. Girls experiencing greater peer chronic stress were the most likely to develop depression.

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We report on a computational investigation of the passive transport of H2 and O2 between the external solution and the hydrogen-producing active site of CpI [FeFe]-hydrogenase from Clostridium pasteurianum. Two distinct methodologies for studying gas access are discussed and applied: (1) temperature-controlled locally enhanced sampling, and (2) volumetric solvent accessibility maps, providing consistent results. Both methodologies confirm the existence and function of a previously hypothesized pathway and reveal a second major pathway that had not been detected by previous analyses of CpI's static crystal structure. Our results suggest that small hydrophobic molecules, such as H2 and O2, diffusing inside CpI, take advantage of well-defined preexisting packing defects that are not always apparent from the protein's static structure, but that can be predicted from the protein's dynamical motion. Finally, we describe two contrasting modes of intraprotein transport for H2 and O2, which in our model are differentiated only by their size.

The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O 2 free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O 2 migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k cat /K m (O 2 ), supporting multiple dynamic pathways for molecular oxygen to reach the active site.Copper amine oxidases are ubiquitous copper containing enzymes that oxidize primary amines to aldehydes through the reduction of molecular oxygen to hydrogen peroxide. CAO 2 catalysis is dependent upon the protein-derived cofactor 2,4,5-trihydroxyphenylalaninequinone (TPQ). The TPQ is derived from an endogenous tyrosine through a self-catalytic process requiring only molecular oxygen and Cu(II) (see Fig. 1a) (1).Hansenula polymorpha 3 amine oxidase is the eukaryotic CAO that has been kinetically characterized in the most detail (2-7). HPAO follows a Bi Bi ping-pong reaction mechanism that can be expressed as two half-reactions, reductive and oxidative (see Fig. 1b). In the reductive half-reaction the enzyme oxidizes a primary amine to an aldehyde, generating the 2e Ϫ reduced aminoquinol form of the cofactor. In the subsequent oxidative half-reaction molecular oxygen is reduced to hydrogen peroxide via cofactor reoxidation to TPQ. Biochemical studies from several different laboratories have led to mechanistic proposals for the catalytic cycle of CAOs (8, 9). These studies have given significant insight into the mechanism for the reductive half-reaction (10). However, the details surrounding the activation of molecular oxygen both in terms of the biogenesis of the TPQ (11, 12) and of the catalytic oxidative half-reaction, remain the subject of intense study (13-16). The utilization of copper as a redox center has been the focus of recent controversy. Because CAOs contain a copper ion in their active site, chemical intuition suggests Cu(I) as the O 2 -activating species to give Cu(II)-superoxide (17). Upon a...