Michael E. P. Murphy

Raster3D Version 2.0. A program for photorealistic molecular graphics.

Raster3D Version 2.0 is a program suite for the production of photorealistic molecular graphics images. The code is hardware independent, and is particularly suited for use in producing large raster images of macromolecules for output to a film recorder or high-quality color printer. The Raster3D suite contains programs for composing illustrations of space-filling models, ball-and-stick models and ribbon-and-cylinder representations. It may also be used to render figures composed using other graphics tools, notably the widely used program Molscript [Kraulis (1991). J. Appl. Cryst. 24, 946-950].

[24] Nitric oxide assay using hemoglobin method

Publisher Summary Hemoglobin, along with related hemoproteins such as myoglobin, can be used for the measurement of nitric oxide (NO). The technique is based on the direct reaction between NO and the oxygenated, ferrous form of hemoglobin (HbO 2 ), which yields the ferric form, methemoglobin (metHb), and nitrate. This technique has the following advantages over other methods of NO measurement: all reagents are inexpensive and readily available, no complex techniques are required for sample collection or handling, the only large equipment needed for the basic technique is a spectrophotometer, and the assay is conducted under physiological conditions. This chapter discusses the basic method and describes the applications of the method to a variety of model systems, and points out possible interferences and artifacts. The chapter discusses the reaction that is the basis for the use of hemoproteins as efficient agents to block the biological effects of NO (the endothelium-derived relaxing factor, EDRF). This reaction is also proposed as a basis for the measurement of NO, using electron spin resonance (ESR) to detect and quantify the nitrosylhemoprotein product.

Ferritin is used for iron storage in bloom-forming marine pennate diatoms

Primary productivity in 30–40% of the world’s oceans is limited by availability of the micronutrient iron. Regions with chronically low iron concentrations are sporadically pulsed with new iron inputs by way of dust or lateral advection from continental margins. Addition of iron to surface waters in these areas induces massive phytoplankton blooms dominated primarily by pennate diatoms. Here we provide evidence that the bloom-forming pennate diatoms Pseudo-nitzschia and Fragilariopsis use the iron-concentrating protein, ferritin, to safely store iron. Ferritin has not been reported previously in any member of the Stramenopiles, a diverse eukaryotic lineage that includes unicellular algae, macroalgae and plant parasites. Phylogenetic analyses suggest that ferritin may have arisen in this small subset of diatoms through a lateral gene transfer. The crystal structure and functional assays of recombinant ferritin derived from Pseudo-nitzschia multiseries reveal a maxi-ferritin that exhibits ferroxidase activity and binds iron. The protein is predicted to be targeted to the chloroplast to control the distribution and storage of iron for proper functioning of the photosynthetic machinery. Abundance of Pseudo-nitzschia ferritin transcripts is regulated by iron nutritional status, and is closely tied to the loss and recovery of photosynthetic competence. Enhanced iron storage with ferritin allows the oceanic diatom Pseudo-nitzschia granii to undergo several more cell divisions in the absence of iron than the comparably sized, oceanic centric diatom Thalassiosira oceanica. Ferritin in pennate diatoms probably contributes to their success in chronically low-iron regions that receive intermittent iron inputs, and provides an explanation for the importance of these organisms in regulating oceanic CO2 over geological timescales.

Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on α-tocopherol

The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.

Structure of nitrite bound to copper-containing nitrite reductase from Alcaligenes faecalis. Mechanistic implications.

The structures of oxidized, reduced, nitrite-soaked oxidized and nitrite-soaked reduced nitrite reductase from Alcaligenes faecalis have been determined at 1.8–2.0 Å resolution using data collected at −160 °C. The active site at cryogenic temperature, as at room temperature, contains a tetrahedral type II copper site liganded by three histidines and a water molecule. The solvent site is empty when crystals are reduced with ascorbate. A fully occupied oxygen-coordinate nitrite occupies the solvent site in crystals soaked in nitrite. Ascorbate-reduced crystals soaked in a glycerol-methanol solution and nitrite at −40 °C remain colorless at −160 °C but turn amber-brown when warmed, suggesting that NO is released. Nitrite is found at one-half occupancy. Five new solvent sites in the oxidized nitrite bound form exhibit defined but different occupancies in the other three forms. These results support a previously proposed mechanism by which nitrite is bound primarily by a single oxygen atom that is protonable, and after reduction and cleavage of that N–O bond, NO is released leaving the oxygen atom bound to the Cu site as hydroxide or water.

Side-On Copper-Nitrosyl Coordination by Nitrite Reductase

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant from the copper cofactor. Comparison of this structure with a refined nitrite-bound crystal structure explains how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The side-on copper-nitrosyl in nitrite reductase expands the possibilities for nitric oxide interactions in copper proteins such as superoxide dismutase and prions.

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem‐iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram‐positive bacteria. Many NEAT domain‐containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem‐binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo‐IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter‐like β‐sandwich fold with a large hydrophobic haem‐binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co‐ordinated solely by Tyr166. The phenol groups of Tyr166 and Tyr170 form an H‐bond that may function in regulating haem binding and release. An analysis of IsdA structure‐sequence alignments indicate that conservation of Tyr166 is a predictor of haem binding by NEAT domains.

Structural biology of heme binding in the Staphylococcus aureus Isd system

Iron is an absolute requirement for nearly all organisms, but most bacterial pathogens are faced with extreme iron-restriction within their host environments. To overcome iron limitation pathogens have evolved precise mechanisms to steal iron from host supplies. Staphylococcus aureus employs the iron-responsive surface determinant (Isd) system as its primary heme-iron uptake pathway. Hemoglobin or hemoglobin-haptoglobin complexes are bound by Near iron-Transport (NEAT) domains within cell surface anchored proteins IsdB or IsdH. Heme is stripped from the host proteins and transferred between NEAT domains through IsdA and IsdC to the membrane transporter IsdEF for internalization. Once internalized, heme can be degraded by IsdG or IsdI, thereby liberating iron for the organism. Most components of the Isd system have been structurally characterized to provide insight into the mechanisms of heme binding and transport. This review summarizes recent research on the Isd system with a focus on the structural biology of heme recognition.

[63] Visible-range low-level chemiluminescence in biological systems

Publisher Summary This chapter discusses the application of visible-range low-level chemiluminescence in biological systems. Two photoemissive processes, fluorescence and phosphorescence, can follow excitation and are collectively termed low-level chemiluminescence or bioluminescence. Singlet oxygen and triplet carbonyls are likely to account for most of the chemiluminescence in biological systems, but it should be mentioned that the formation and emission from excited species probably represent only minor side reactions of the biological pathways that act as their source. Highly sensitive photodetectors can measure the low-level (or ultraweak) chemiluminescence accompanying these processes even in complex biological systems. The monitoring of this low-level chemiluminescence has been used together with other methods of assessing oxidative processes within cells, but it has the advantages of being noninvasive and providing continuous monitoring. In addition to the procedures for the direct measurement of ongoing chemiluminescence, the specific enhancers and quenchers of activated compounds can aid in the identification of the luminescent species in some biological systems.

Type-2 copper-containing enzymes.

Abstract.Type-2 Cu sites are found in all the major branches of life and are often involved in the catalysis of oxygen species. Four type-2 Cu protein families are selected as model systems for review: amine oxidases, Cu monooxygenases, nitrite reductase/multicopper oxidase, and CuZn superoxide dismutase. For each model protein, the availability of multiple crystal structures and detailed enzymological studies provides a detailed molecular view of the type-2 Cu site and delineation of the mechanistic role of the Cu in biological function. Comparison of these model proteins leads to the identification of common properties of the Cu sites and insight into the evolution of the trinuclear active site found in multicopper oxidases.