Thomas L. Poulos

Bio: Thomas L. Poulos is an academic researcher from University of California, Irvine. The author has contributed to research in topics: Heme & Cytochrome c peroxidase. The author has an hindex of 78, co-authored 356 publications receiving 26423 citations. Previous affiliations of Thomas L. Poulos include University of California & Vanderbilt University Medical Center.

Heme Enzyme Structure and Function

Abstract: Metalloporphyrins are widely used throughout the biosphere and of these heme (iron protoporphyrin IX, Fig. 1) is one of the most abundant and widely used. Heme shuttles electrons between proteins as in mitochondrial respiration or transports and stores O2 as with the globins. The role of heme in more active enzymatic chemical transformation began to be appreciated just after the discovery by Mason1 and Hayaishi2 that O2 O atoms can be enzymatically incorporated into organic substrates which represented the seminal discovery of oxygenases. While the enzymes used in these studies did not contain heme, it was not too long before heme-containing oxygenases also were discovered. In 1958 Klingenberg3 and Garfinkel4 found an unusual pigment in microsomes that when reduced in the presence of CO generated a spectrum with a peak at 450 nm instead of the expected 420 nm peak. Hence the name P450 was born. In 1964 Omura and Sato5,6 showed that this “pigment” is actually a protein and the function of this strange heme protein became clear in a seminal study by Estabrook et al.7 that demonstrated the involvement of the 450 nm pigment in steroid hydroxylation. Thus by the mid-1960s it was established that heme plays an active role in biology by somehow catalyzing the hydroxylation of organic substrates. While these discoveries certainly mark the beginning of modern approaches to studying heme enzyme oxygenases, the enzymatic role of heme dates much earlier to 1903 when horseradish peroxidase (HRP) was described.8 Indeed, owing to the ease of purification and stability of the various intermediates, HRP dominated heme enzyme studies until P450 was discovered. Figure 1 Structure of iron protoporphyrin IX. Heme enzymes can catalyze both reductive and oxidative chemistry but here we focus on those that catalyze oxidation reactions, and especially those for which crystal structures are available. There are two broad classes of heme enzyme oxidants: oxygenases that use O2 to oxidize, usually oxygenate, substrates and peroxidases that use H2O2 to oxidize, but not normally oxygenate, substrates. Of the two oxidants molecular oxygen is the most unusual because even though the oxidation of nearly all biological molecules by O2 is a thermodynamically favorable process, O2 is not a reactive molecule. The reason, of course, is that there is a large kinetic barrier to these reactions owing to O2 being a paramagnetic molecule so the reaction between a majority of biological molecules that have paired spins is a spin forbidden process. Overcoming this barrier is why Nature recruited transition metals and heme into enzyme active sites. As shown in Fig. 2, heme oxygenases bind O2 and store the O2 oxidizing equivalents in the iron, porphyrin, and/or amino acid side chains for further selective oxidation of substrates. Peroxidases use H2O2 as the oxidant and while not having the O2 spin barrier, H2O2 presents its own problems. The reaction between H2O2 and transition metals generates toxic hydroxyl radicals in the well known Fenton chemistry9 which would be highly destructive to enzyme active sites. As illustrated in Fig. 2, all heme oxidases are at some point in the catalytic cycle peroxidases. Molecular oxygen must first be reduced by two electrons to the peroxide level before the interesting chemistry starts: cleavage of the O-O bond. This bond can cleave either homolytically, which gives two hydroxyl radicals, or heterolytically to effectively give H2O and a naked O atom with only 6 valence electrons. Since the release of hydroxyl radicals in the active site must, in most cases, be avoided Nature has engineered heme enzyme active sites to ensure that the heterolytic pathway dominates. Figure 2 Oxygen and peroxide activation by heme enzymes. Oxygenases like P450 must have the iron reduced to ferrous (Fe(II) or Fe2+) before O2 can bind. The oxy complex is best described as ferric-superoxide, Fe(III)-OO−. A second electron transfer results ... The list of heme enzymes is substantial and thus it is necessary to be selective on which to discuss in detail. It may appear that a disproportionate amount of space is devoted to peroxidases and P450s. This is true and admittedly reflects the author’s own interests and area of expertise. Additionally, however, peroxidases are the most extensively studied heme enzymes and have provided fundamental insights into the chemistry and structure shared by many other enzymes. The other enzymes to be discussed were selected owing to both subtle variations on common themes and novel features that Nature selected for specific biological function.

Crystal structure of horseradish peroxidase C at 2.15 A resolution.

Abstract: The crystal structure of horseradish peroxidase isozyme C (HRPC) has been solved to 2.15 A resolution. An important feature unique to the class III peroxidases is a long insertion, 34 residues in HRPC, between helices F and G. This region, which defines part of the substrate access channel, is not present in the core conserved fold typical of peroxidases from classes I and II. Comparison of HRPC and peanut peroxidase (PNP), the only other class III (higher plant) peroxidase for which an X-ray structure has been completed, reveals that the structure in this region is highly variable even within class III. For peroxidases of the HRPC type, characterized by a larger FG insertion (seven residues relative to PNP) and a shorter F′ helix, we have identified the key residue involved in direct interactions with aromatic donor molecules. HRPC is unique in having a ring of three peripheral Phe residues, 142, 68 and 179. These guard the entrance to the exposed haem edge. We predict that this aromatic region is important for the ability of HRPC to bind aromatic substrates.

Processing of X-ray diffraction data collected in oscillation mode

Abstract: Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.

Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function

Abstract: A novel and robust automated docking method that predicts the bound conformations of flexible ligands to macromolecular targets has been developed and tested, in combination with a new scoring function that estimates the free energy change upon binding. Interestingly, this method applies a Lamarckian model of genetics, in which environmental adaptations of an individual's phenotype are reverse transcribed into its genotype and become . heritable traits sic . We consider three search methods, Monte Carlo simulated annealing, a traditional genetic algorithm, and the Lamarckian genetic algorithm, and compare their performance in dockings of seven protein)ligand test systems having known three-dimensional structure. We show that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckian genetic algorithm is the most efficient, reliable, and successful of the three. The empirical free energy function was calibrated using a set of 30 structurally known protein)ligand complexes with experimentally determined binding constants. Linear regression analysis of the observed binding constants in terms of a wide variety of structure-derived molecular properties was performed. The final model had a residual standard y1 y1 .

Reactive Oxygen Species, Oxidative Damage, and Antioxidative Defense Mechanism in Plants under Stressful Conditions

Abstract: Reactive oxygen species (ROS) are produced as a normal product of plant cellular metabolism. Various environmental stresses lead to excessive production of ROS causing progressive oxidative damage and ultimately cell death. Despite their destructive activity, they are well-described second messengers in a variety of cellular processes, including conferment of tolerance to various environmental stresses. Whether ROS would serve as signaling molecules or could cause oxidative damage to the tissues depends on the delicate equilibrium between ROS production, and their scavenging. Efficient scavenging of ROS produced during various environmental stresses requires the action of several nonenzymatic as well as enzymatic antioxidants present in the tissues. In this paper, we describe the generation, sites of production and role of ROS as messenger molecules as well as inducers of oxidative damage. Further, the antioxidative defense mechanisms operating in the cells for scavenging of ROS overproduced under various stressful conditions of the environment have been discussed in detail.