Heme binding to human CLOCK affects interactions with the E-box.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

In-Situ Estimation of Soil Water Retention Curve in Silt Loam and Loamy Sand Soils at Different Soil Depths.

The soil water retention curve (SWRC) shows the relationship between soil water (θ) and water potential (ψ) and provides fundamental information for quantifying and modeling soil water entry, storage, flow, and groundwater recharge processes. While traditionally it is measured in a laboratory through cumbersome and time-intensive methods, soil sensors measuring in-situ θ and ψ show strong potential to estimate in-situ SWRC. The objective of this study was to estimate in-situ SWRC at different depths under two different soil types by integrating measured θ and ψ using two commercial sensors: time-domain reflectometer (TDR) and dielectric field water potential (e.g., MPS-6) principles. Parametric models were used to quantify θ-ψ relationships at various depths and were compared to laboratory-measured SWRC. The results of the study show that combining TDR and MPS-6 sensors can be used to estimate plant-available water and SWRC, with a mean difference of -0.03 to 0.23 m3m-3 between the modeled data and laboratory data, which could be caused by the sensors' lack of site-specific calibration or possible air entrapment of field soil. However, consistent trends (with magnitude differences) indicated the potential to use these sensors in estimating in-situ and dynamic SWRC at depths and provided a way forward in overcoming resource-intensive laboratory measurements.

Structural and spectroscopic characterisation of a heme peroxidase from sorghum.

A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions.

Crystal structure of reduced MsAcg, a putative nitroreductase from Mycobacterium smegmatis and a close homologue of Mycobacterium tuberculosis Acg.

This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria's adaptation to a dormancy state.

Bacterial contamination of tile drainage water and shallow groundwater under different application methods of liquid swine manure.

A 2 year field experiment evaluated liquid manure application methods on the movement of manure-borne pathogens (Salmonella sp.) and indicator bacteria (Escherichia coli and Clostridium perfringens) to subsurface water. A combination of application methods including surface application, pre-application tillage, and post-application incorporation were applied in a randomized complete block design on an instrumented field site in spring 2007 and 2008. Tile and shallow groundwater were sampled immediately after manure application and after rainfall events. Bacterial enumeration from water samples showed that the surface-applied manure resulted in the highest concentration of E. coli in tile drainage water. Pre-tillage significantly (p < 0.05) reduced the movement of manure-based E. coli and C. perfringens to tile water and to shallow groundwater within 3 days after manure application (DAM) in 2008 and within 10 DAM in 2007. Pre-tillage also decreased the occurrence of Salmonella sp. in tile water samples. Indicator bacteria and pathogens reached nondetectable levels within 50 DAM. The results suggest that tillage before application of liquid swine manure can minimize the movement of bacteria to tile and groundwater, but is effective only for the drainage events immediately after manure application or initial rainfall-associated drainage flows. Furthermore, the study highlights the strong association between bacterial concentrations in subsurface waters and rainfall timing and volume after manure application.

A Bio-Economic Crop Yield Response (BECYR) Model for Corn and Soybeans in Ontario, Canada for 1959-2013.

This paper presents estimates of the effects of changing climate on crop yields for grain corn and soybeans in Ontario, Canada, for 1959-2013. We were able to use a database that is more comprehensive with respect to explanatory variables than some previous efforts had available. Our model includes climate variables, prices, land quality, groundwater level, CO2 concentration, and a time trend. Our results indicate that trends in temperature and precipitation during our study period have not yet resulted in appreciable threats to crop yields in the region.

A simple method for the determination of reduction potentials in heme proteins.

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (Em=-115 mV ± 2 mV, pH 7.0) and human CLOCK (Em=-111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.