Search

Heme nitric oxide/oxygen (H-NOX) proteins are found in eukaryotes where they are typically part of a larger protein such as soluble guanylate cyclase and in prokaryotes where they are often found in operons with a histidine kinase, suggesting that H-NOX proteins serve as sensors for NO and [O.sub.2] in signaling pathways. The Fe(II)-NO complex of the H-NOX protein from Shewanella oneidensis inhibits the autophosphorylation of the operon-associated histidine kinase, whereas the ligand-free H-NOX has no effect on the kinase. NMR spectroscopy was used to determine the structures of the Fe(II)-CO complex of the S. oneidensis H-NOX and the Fe(II)-CO complex of the H103G H-NOX mutant as a mimic of the ligand-free and kinaseinhibitory Fe(II)-NO H-NOX, respectively. The results provide a molecular glimpse into the ligand-induced conformational changes that may underlie kinase inhibition and the subsequent control of downstream signaling. hemoprotein | nitric oxide | signaling | NMR | phosphorylation doi/10.1073/pnas.0911645106

Cytochrome P450s form a large and diverse family of heme-containing proteins capable of carrying out many different enzymatic reactions. In both mammals and plants, some P450s are known to carry out reactions essential for processes such as hormone synthesis, while other P450s are involved in the detoxification of environmental compounds. In general, functions of insect P450s are less well understood. We characterized Drosophila melanogaster P450 expression patterns in embryos and 2 stages of third instar larvae. We identified numerous P450s expressed in the fat body, Malpighian (renal) tubules, and in distinct regions of the midgut, consistent with hypothesized roles in detoxification processes, and other P450s expressed in organs such as the gonads, corpora allata, oenocytes, hindgut, and brain. Combining expression pattern data with an RNA interference lethality screen of individual P450s, we identify candidate P450s essential for developmental processes and distinguish them from P450s with potential functions in detoxification. detoxification genes | in situ hybridization | insecticide resistance | multigene family | RNAi

We have found that in the presence of hydroxylamine, the heme prosthetic group of the heme protein adsorbed at the mercury electrode surface reacts with mercury ion produced by the electrochemical oxidation of mercury and is quantitatively converted into the mercury protoporphyfin IX group using single-sweep polarography. As a result, the small redox peak [P.sub.0] of the heme prosthetic group at about -0.46 V (vs SCE) disappears and a large new reduction peak P of mercury protoporphyrin IX group at -0.89 V comes out in a pH 9.6 NaHC[O.sub.3]--[Na.sub.2]C[O.sub.3] solution. Peak P is extremely sensitive to heine protein concentration. On the basis of the reduction peak P, a unique electrochemical method for heme protein assays is constructed. For the cytochrome c determination, the peak height is linearly proportional to the concentration in the range of 0.005-15 mg [L.sup.-1] (correlation coefficient 0.999). The detection limit is 0.003 mg [L.sup.-1]. In contrast with peak [P.sub.0], the detection limit of cytochrome c is only 0.6 mg [L.sup.-1]. The voltammograms of heme proteins in the absence and presence of hydroxylamine can serve as a reliable qualitative analytical method. The chemical reaction is peculiar to the heme prosthetic group. Without hydroxylamine it cannot occur. Thereby the method is highly specific and free from interference. The performance takes only a few minutes. These advantages make the method attractive for heme protein detecting.

Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 A from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor. heme binding | iron uptake | membrane protein | membrane transport | protein complex

Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11 E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between [beta]3-2 and [beta]3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism. oxylipin | jasmonate | guayule

Nitric oxide (NO) has been shown to be the endothelium-derived relaxing factor (EDRF), and its impairment contributes to a variety of cardiovascular disorders. Recently, it has been recognized that nitrite can be an important source of NO; however, questions remain regarding the activity and mechanisms of nitrite bioactivation in vessels and its physiological importance. Therefore, we investigated the effects of nitrite on in vivo hemodynamics in rats and in vitro vasorelaxation in isolated rat aorta under aerobic conditions. Studies were performed to determine the mechanisms by which nitrite is converted to NO. In anesthetized rats, nitrite dose dependently decreased both systolic and diastolic blood pressure with a threshold dose of l0 [micro]M. Similarly, nitrite (10 [micro]M-2 mM) caused vasorelaxation of aortic rings, and NO was shown to be the intermediate factor responsible for this activity. With the use of electrochemical as well as electron paramagnetic resonance (EPR) spectroscopy techniques NO generation was measured from isolated aortic vessels following nitrite treatment. Reduction of nitrite to NO was blocked by heating the vessel, suggesting that an enzymatic process is involved. Organ chamber experiments demonstrated that aortic relaxation induced by nitrite could be blocked by both hemoglobin and soluble guanylyl cyclase (sGC) inhibitor 1H[1,2,4] oxadiazolo[4,3-a]quinoxaline-l-one (ODQ). In addition, both electrochemical and EPR spin-trapping measurements showed that ODQ inhibits nitrite-mediated NO production. These findings thus suggest that nitrite can be a precursor of EDRF and that sGC or other heme proteins inhibited by ODQ catalyze the reduction of nitrite to NO. soluble guanylate cyclase; aerobic nitrite reduction; vasorelaxation; blood pressure; heme proteins

Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to [approximately equal to] 100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the protein's structure. FTIR | neuroglobin | ultrafast 2D-IR

Biological transformations of polyunsaturated fatty acids often lead to chemically unstable products, such as the prostaglandin endoperoxides and leukotriene [A.sub.4] epoxide of mammalian biology and the allene epoxides of plants. Here, we report on the enzymatic production of a fatty acid containing a highly strained bicyclic four-carbon ring, a moiety known previously only as a model compound for mechanistic studies in chemistry. Starting from linolenic acid (C18.3[omega]3), a dual function protein from the cyanobacterium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.3[omega]3 in a lipoxygenase domain, then a catalase-related domain converts the 9R-hydroperoxide to two unstable allylic epoxides. We isolated and identified the major product as 9R, 10R-epoxy-11trans-C18.1 containing a bicyclo[1.1.0]butyl ring on carbons 13-16, and the minor product as 9R, 10R-epoxy-11trans,13trans,15cis-C18.[omega]3, an epoxide of the leukotriene A type. Synthesis of both epoxides can be understood by initial transformation of the hydroperoxide to an epoxy allylic carbocation. Rearrangement to an intermediate bicyclobutonium ion followed by deprotonation gives the bicyclobutane fatty acid. This enzymatic reaction has no parallel in aqueous or organic solvent, where ring-opened cyclopropanes, cyclobutanes, and homoallyl products are formed. Given the capability shown here for enzymatic formation of the highly strained and unstable bicyclobutane, our findings suggest that other transformations involving carbocation rearrangement, in both chemistry and biology, should be examined for the production of the high energy bicyclobutanes. catalase | carbocation | epoxide | leukotriene | bicyclobutonium ion

Neuroglobin (Ngb), a protein in the globin family, is found in vertebrate brains. It binds oxygen reversibly. Compared with myoglobin (Mb), the amino acid sequence has limited similarity, but key residues around the heme and the classical globin fold are conserved in Ngb. The CO adduct of Ngb displays two CO absorption bands in the IR spectrum, referred to as [N.sub.3] (distal histidine in the pocket) and [N.sub.o] (distal histidine swung out of the pocket), which have absorption spectra that are almost identical with the Mb mutants L29F and H64V, respectively. The Mb mutants mimic the heme pocket structures of the corresponding Ngb conformers. The equilibrium protein dynamics for the CO adduct of Ngb are investigated by using ultrafast 2D-IR vibrational echo spectroscopy by observing the CO vibration's spectral diffusion (2D-IR spectra time dependence) and comparing the results with those for the Mb mutants. Although the heme pocket structure and the CO FTIR peak positions of Ngb are similar to those of the mutant Mb proteins, the 2D-IR results demonstrate that the fast structural fluctuations of Ngb are significantly slower than those of the mutant Mbs. The results may also provide some insights into the nature of the energy landscape in the vicinity of the folded protein free energy minimum. myoglobin mutants | protein dynamics | energy landscape

The site directed nitroxide spin labeling (SDSL) of cysteine residues were used to search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome b[o.sub.3], Escherichia coli. The kinetic studies of mutants demonstrate that the redox gating of protons occurs deep within the D channel close to the heme-copper site implies that no motion is transmitted to the ends of the helices.

The flow of photoexcitation energy in biomolecules is investigated. It is demonstrated that a dynamically evolving absorption band leads directly to a time dependent complex line shape function and REP, which must be considered along with the Bose-Einstein factor when attempting to extract a time dependent vibrational temperature from picosecond resonance raman data.

The binding of heme to NikA and the way in which mutation of NikA can diminish the production of P-574 in anaeorobic CydDC-overexpressing cells is described. The expression patterns of NikA and cytochrome bd have shown that the data is consistent with a link between the CydDC transporter, NikA, and heme processing during oxidase assembly.

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1 was dominant is described. The findings suggest that purified enzyme might be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.

An ab initio study, in combination with the density functional theory (DFT) is used to analyze the potential energy surfaces (PESs) and the associated energy barriers, characterizing the spin-forbidden recombination reactions of various deoxy-heme groups with several ligands. The results provide a complete set of values of bond energies for such ligands and show that even though water does not involve a barrier, it cannot be used as a binding element for heme proteins.

Multi-Conformation Continuum Electrostatics (MCCE) was used to calculate proton uptake during the individual stages of anaerobic reduction of Rhodobacter sphaeroides cytochrome c oxidase. The propionic acids near the binuclear center (BNC) are deprotonted with p[K.sub.a]S well below 7 and are well stabilized in their anionic state and do not bind a proton upon BNC reduction which suggests that electroneutrality in the BNC is not maintained during reduction.

Structural characterization of the spontaneous 'aging' of the heme oxygenase from Neisseria meningitidis, NmHO through cleavage of the C-terminus in contact with the substrate is presented. The cleavage occurs over a wide range of rates depending on the nature of the substrate and axial ligand and is not inhibited in the absence of O2 or in the presence of protease inhibitor cocktail.