Search

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming ( time-resolved Raman spectroscopy | allostery | structural dynamics doi/ 10.1073/pnas.0912938107

The surface oxidation site (Trp-171) in lignin peroxidase (LIP) required for the reaction with veratryl alcohol a high-redox-potential (1.4 V) substrate, was engineered into Coprinus cinereus peroxidase (CiP) by introducing a Trp residue into a heme peroxidase that has similar protein fold but lacks this activity. To create the catalytic activity toward veratryl alcohol in CiP, it was necessary to reproduce the Trp site and its negatively charged microenvironment by means of a triple mutation. The resulting D179W+R258E+R272D variant was characterized by multifrequency EPR spectroscopy. The spectra unequivocally showed that a new Trp radical [g values of [g.sub.x] = 2.0035(5), [g.sub.y] = 2.0027(5), and [g.sub.z] = 2.0022(1)] was formed after the [Fe(IV)=O [Por.sup.*+]] intermediate, as a result of intramolecular electron transfer between Trp-179 and the porphyrin. Also, the EPR characterization crucially showed that [Fe(IV=O Trp-179[sup.*]] was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment, as well as those of the preceding [Fe(IV)-O [Por.sup.*+]] intermediate to engineer a catalytically competent Trp site for a given substrate. Manipulation of the microenvironment of the Trp-171 site in LiP allowed the detection by EPR spectroscopy of the Trp-171[sup.*], for which direct evidence has been missing so far. Our work also highlights the role of Trp residues as tunable redox-active cofactors for enzyme catalysis in the context of peroxidases with a unique reactivity toward recalcitrant substrates that require oxidation potentials not realized at the heme site. heme peroxidase | high-field EPR spectroscopy | protein engineering | tryptophan radical | electron transfer

The recombinant expression and initial characterization of the putative human enteric ferric reductase Dcytb is examined. The characterization of the wild type and variant forms of the protein provides evidence concerning the number of heme binding sites and identification of the axial ligands.

The comparative studies of binding of NO and CO to chelated protoheme-L-histidine methyl ester (HM-H), protoheme-glycyl-L-histidine methyl ester (HM-Gh), and free protoheme (HM) is reported.

High-pressure spectroscopy is used for studying allosteric mechanisms in human cytochrome P450 3A4 (CYP3A4) in oligomers in solution or monomeric enzyme incorporated into Nanodiscs (CYP3A4ND). The results have indicated that the mechanisms of interactions of CYP3A4 with 1-pyrenebuanol (1-PB) and testosterone have involved an effector-induced transition.

The study is done to determine the heme coordination states of inducible nitric oxide synthase with a Re (I) sensitizer coupled to nitro-L-arginine by an imidazole-alkyl molecular wire. It is demonstrated that Re (I) when applied can initiate quick photoreduction of the ferriheme in the active site of the enzyme.

Heme degradation pathway in the mosquito Aedes aegypti is described. It is found that in contrast to the Rhodnius prolixus, Aedes aegypti involves oxidative cleavage of heme without previous modification in the [alpha] meso-carbon.

The syntheses and characterization of the iron(II) complexes of the pentadentate amide-containing macrocyclic ligand 3,6,9,12,18-pentaazabicyclo-[12,3,1]octadeca-1(18),14,16-triene-2,13-dione ([H.sub.2]pydione[N.sub.5]) are reported. The complexes maintain a high-spin state even at low temperature and detailed kinetic studies of oxygenation of the iron(II) complexes showed that the deprotonation state of the complex has a profound effect on the reactivity with dioxygen.

The formation of a dioxygen adduct, [([F.sub.8]TPP)F[e.sup.III]-([O.sub.2.sup.2-])-C[u.sup.II](TMPA)](Cl[O.sub.4]) obtained by addition of oxygen to a 1:1 mixture of the porphyrinate-iron(II) complex ([F.sub.8]TPP)F[e.sup.II] {[F.sub.8]TPP = tetrakis-(2,6-difluorophenyl)porphyrinate dianion} and the copper(I) complex [(TMPA)C[u.sup.1](MeCN)](Cl[O.sub.4]) {TMPA = tris-(2-pyridylmethyl)amine} is described. Thermal decomposition of [mu]-peroxo complex [([F.sub.8]TPP)F[e.sup.III]-([O.sub.2.sup.2-])-C[u.sup.II](TMPA)](Cl[O.sub.4]) yields [mu]-oxo complex [[([F.sub.8]TPP)F[e.sup.III]-O-C[u.sup.II](TMPA)].sup.+] with concomitant release of ~0.5 mol oxygen per mol and provides new insights to heme/Cu dioxygen-binding and reduction.

Quinol/nitrate oxidoreductase (NarGHI) complex in Escherichia coli is known to operate with ubi and menaquinones however the location and the number of quinol binding sites remain indefinable. So a study is conducted to identify its location and function using a combination of mutagenesis, kinetics, EPR and ENDOR spectroscopies.

Various stopped-flow investigations are conducted to study the proton-directed redox control of O-O bond activation by the models of several heme cofactor of hydroperoxidase enzymes. The results show that the Hangman metalloporphyrin complexes exhibit an exceptional catalytic performance, which proves that such proton-coupled multielectron reactions can be used to catalyze O-O bond breaking reactions.

The identification of a conserved Mc [b.sub.5] apoprotein (core 2) packing motif that plays a key role in stabilizing apoprotein conformation in the vicinity of Trp-22 is reported. Evidence indicates that the conserved Mc [b.sub.5] packing motif noted above contributes to the unusually extensive secondary structure exhibited by bovine Mc apo-[b.sub.5] in the area-denatured state.

A study on the responses of the catalytic center to external perturbations is studied and density functional theory calculations on an active-site model of catalase compound I are conducted. Results reveal that oxo-iron heme systems are chameleonic species that are influenced by external perturbations and change their character and catalytic properties depending on the local environment.

A study presents the biophysical characterization of the sterol demethylase P450 (CYP51), which is obtained from Mycobacterium tuberculosis (MtB) and is chromosomally located adjacent to a ferrodoxin (Fdx). The analysis reveals that despite a thermodynamic barrier, Fdx forms a productive electron transfer complex and reduces it to a specific rate, as heme iron reduction is found to act as a rate-limiting step.

HemAT from Bacillus subtilis (HemAT-Bs) is a commonly used heme-based [O.sub.2] sensor protein which consists of various specific hydrogen-bonding networks and is often used as a signal transducer responsible for aerotaxis. The various experiments and techniques used show that the formation of hydrogen bonds induces a conformational change in the E-helix, which is extremely essential for oxygen sensing and signal transduction in the protein.

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states is investigated by using experimental and computational methods. Fluorescence emission experiments and computational docking simulations have shown that the polarity probe NR binds to ApoMb within the heme binding pocket.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.02.003 Byline: Stephen P.L. Cary (1), Jonathan A. Winger (2), Emily R. Derbyshire (3), Michael A. Marletta (1)(3)(4) Abstract: Nitric oxide (NO) triggers various physiological responses in numerous tissues by binding and activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. In vivo, basal NO/cGMP signaling maintains a resting state in target cells (for example, resting tone in smooth muscle), but an acute burst of NO/cGMP signaling triggers rapid responses (such as smooth muscle relaxation). Recent studies have shown that the sGC heterodimer comprises at least four modular domains per subunit. The N-terminal heme domain is a member of the H-NOX family of domains that bind O.sub.2 and/or NO and are conserved in prokaryotes and higher eukaryotes. Studies of these domains have uncovered the molecular basis for ligand discrimination by sGC. Other work has identified two temporally distinct states of sGC activation by NO: formation of a stable NO-heme complex results in a low-activity species, and additional NO produces a transient fully active enzyme. Nucleotides also allosterically modulate the duration and intensity of enzyme activity. Together, these studies suggest a biochemical basis for the two distinct types of NO/cGMP signal observed in vivo. Author Affiliation: (a) Department of Chemistry (b) Howard Hughes Medical Institute (c) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA (d) Division of Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

The kinetic implications and energy profile in a roller coaster electron transfer chain are examined, based on a study of modified tetraheme-reaction center constructs. The energy-profile of the electron transfer chain presented an additional uphill step either downstream or upstream from c559.

Spectrally resolved infrared vibrational echo spectroscopy(super 60-63) is used to measure the fast dynamics of heme-bound CO in MbCO and HbCO embedded in silica sol-gel glasses. IR vibrational spectrosocpy provides valuable insights into the influence of the sol-gel confinement on protein dynamics.

Reactive studies are performed of the iron-hydroperoxo species in nucleophilic and electrophilic reactions with in situ-generated mononuclear non-heme iron(III)-hydroperoxo complexes that are well characterized with various spectroscopic techniques. Results demonstrate that non-heme iron(III)- hydroperoxo species are sluggish oxidants and that the oxidizing power of the intermediates cannot compete with that of high-valent iron(IV)-oxo complexes.

The applicability of Badger's rule to heme and non-heme iron-oxygen bonds is examined to gain insights into the protonation state of enzymatic ferryl species. The long Fe-O bonds reported in the crystal structures of the ferryl forms of myoglobin, cytochrome c peroxidase and catalase deviate substantially from the values predicted by Badger's rule, while the short Fe-O bonds obtained from X-ray absorption measurements are in good agreement with Badger's rule.

The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. The first comprehensive measurements of the temperature-dependent NO rebinding kinetics of Mb and its distal pocket mutants are presented.

Amphirite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogeneted phenols to the corresponding quinones. It is shown that unlike other peroxidases, DHP activity requires the addition of substrate, 2,4,6-tribromophenol, prior to the cosubstrate, peroxide.

Electron/proton transfer was studied in photosystem II (PSII) and the redox properties of the non-heme iron complex (Fe complex), which are sensitive to the redox state of quinones (Q (sub (A/B)). The result showed forming of Fe(super 3)(super plus)Q(sub B)H(sub 2) by a proton-coupled electron transfer process from the initial state Fe(super 2)(super plus)Q(sub A)(super minus) results in deprotonation of D1-His252.

The study demonstrates the detection of His-heme a3(super 2+)-NO/CU(sub B)(super 1+) species in the reduction of NO to N2O by ba3-oxidase. It is suggested that the reduction of NO to N2O by ba3-oxidase proceeds by the fast binding of the first NO molecule to heme a3 with high-affinity, and the second NO molecule binds to Cu(sub B) with low affinity, producing the temporal co-presence of two NO molecules in the hemecopper center.

A series of iron(ii) bis(triflate) complexes containing tripodal tetradentate nitrogen ligands with pyridine and dimethylamine donors of the type [N(CH2Pyr)(sub 3-n)(CH2CH2NMe2)n] [n=0 (tpa, 1), n=1 (iso-bpmen, 3), n=2 (Me4-benpa, 4), n=3 (Me6-tren, 5)] and the linear tetradentate ligand [(Ch2Pyr)MeN(CH2CH2)NMe(CH2Pyr), (bpmen, 2)] are prepared. The catalytic properties of the complexes for the oxidation of alkanes are evaluated using cyclohexane as the substrate.

Density functional theory calculations are used to evaluate hydrogen bond energies of five- and six-coordinate heme-NO complexes with phenol and imidazole, chosen as models for distal pocket tyrosine and histidine residues. The calculated interaction energies are approximately 2 kcal/mol for phenol and 3-4 kcal/mol for imidazole, which are 2-4 times smaller than the energies calculated for heme-O2 complexes hydrogen-bonding with a distal histidine.

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, where as pa-HO yields predominantly biliverdin IXdelta.

The formation, physical properties and small-molecule (CO-PPh3) reactivities of the heme-peroxo-copper species, [((super 2)L)Fe(super III)-(O(sub 2)(super 2-))-Cu(super II)](super +) (6) is reported. This study shows that the nature of Cu(super I)/O2 interactions can be applied to the study of heme/Cu(super I)/O2 reactions.

Optical spectroscopy, MCD and stopped-flow for the pH sensitivity of spectral features are used to investigate the three forms of cytochrome c oxidase, fully oxidized CcO (CcO-O), oxidized CcO complexed with cyanide (CcO.CN), and mixed valence CcO, in which both heme a3 and Cu(sub B) are reduced and stabilized by carbon monoxide (MV.CO). Glu60 of subunit II, located at the entrance of the proton conducting K-channel, is the proteolytic residue that interacts with both hemes through a hydrogen-bonding network.

X-ray structures at 3.0-3.1 angstrom resolution of the cytochrome b6f complex from the cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii showed the presence of a unique heme, heme x. The only axial ligand associated with this heme is a H2O or OH(super -) that is H-bonded to the propionate of the stormal side heme bn, showing that it is pentacoordinate.

An examination was conducted on the effect of isocyanides as axial ligands on the formation and stability of verdoheme by oxidation. No resonances due to paramagnetic products were detected and the reactions appeared to result only in the formation of the diamagnetic verdohemes, and the products were not susceptible to further oxidation.

Reduction kinetics of hexacoordination in hemoglobin, including human neuroglobin and cytoglobin are compared to myoglobin, soybean leghemoglobin and several mutant proteins. In all cases, bis-histidyl coordination greatly increases the rate of reduction by sodium dithionate when compared to pentacoordinate hemoglobins.

The observation and fast time-scale kinetic determination of a primary dioxygen-copper interaction have been investigated in a study. A novel copper 'flash-and-trap' experiment typically used for hemes is performed, leading to the accurate determination of the second-order rate constant for (ligand)Cu/O2 reaction, a value that exceeds that for synthetic and natural hemes.

(Super 13)C nuclear magnetic resonance (NMR) spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase, where OH(super -1) is used as a model of the OOH (super -1) ligand to investigate the ferric hydroperoxide intermediate in heme catabolism at ambient temperatures. Results suggest that if the ligand field strength of the coordinated OOH(super -1) in heme oxygenase is modulated, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.

Oxidation reactions were studied using a synthetic heme-thiolate (SR complex) in order to ascertain the contributions of multiple intermediates derived from heme-thiolate to the oxygen atom transfer reaction to substrate. The substrate and oxidant interact with each other during the oxygen atom transfer reaction.

The effect of trans thiolate ligation on the coordination nitric oxide in ferric heme nitrosyl complexes as a function of the thiolate donor strength, induced by variation of NH-S(thiolate) hydrogen bonds, is examined. The difference between axial N- and S-donor-coordinated ferric heme nitrosyls is due to a p-trans effect of the coordinated S on the bound NO mediated by an NO [sigma]* orbital.

A scanning tunneling microscopy (STM) is used to study the functional aggregation of heme oxygen-sensing/signaling proteins in solution and microperoxidase (MP)11 is adsorbed on Au(111) surfaces. It is revealed that it is possible to systematically control MP11 aggregation on Au(111), while in the STM images, structural details of both the heme ring and the polypeptide substructure in individual MP11 molecules are observed.

The turnover numbers of the active [Fe(III), Fe(II)-[O.sub.2] and Fe(II)-CO] [Fe(III), Fe(III)-protoporphyrin IX complex, Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO] of YddV from Escherichia coli (Ec) were quantified to determine the critical factors in the regulation of c-di-GMP homeostasis in [O.sub.2] binding and the heme redox switch of YddV. The findings clearly supported the critical role of Tyr in oxygen sensing, and hence modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which might be applicable to other globin-coupled oxygen sensor enzymes.

A lipid blend solution comprised of mono- and diglycerides is deposited onto an aqueous surface to generate synthetic neutral lipid droplets (SNLDs), which might have function other than energy storage molecules in Plasmodium falciparum during the intraerythrocytic stage of the parasite life cycle. The results indicated that neutral lipid structures within the digestive vacuole are not only the location of in vivo hemozoin formation but is also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.

Utilization of two regioisomeric iron corroles [[alpha]-CH- and [gamma]-CH-Fe(cor)] as artificial substrate and cofactor analogues to mammalian, plant, cyanobacterial, and bacterial heme oxygenases is described. The selective reaction for the corrole regioisomer and for plant-type heme oxygenases is the first example of an enzymatic corrole ring opening.

A comprehensive genetic, metabolomic and biochemical study on the catalytic properties of Streptomyces coelicolor cytochrome P450 (P450) 154A1, which has a unique heme orientation in its crystal structure, is described. The biotransformation of dipentaenone by P450 154A1 is an unexpected intramolecular cyclization to a Paterno-Buchi-like product, without oxidation/reduction.

Molecular dynamics (MD) simulations of the R108H mutant of cytochrome P450 2C9 (CYP2C9) were performed to study the range of energetically accessible CYP2C9 conformations and understand the movement of the B-C loop region on CYP2C9 away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. The findings proposed a model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 to demonstrate the enzyme' s ability to regio- and stereospecifically metabolize some ligands while allosterically activating others.

A combined study of X-ray crystallography and theoretical QM/MM calculations were applied to understand the mechanism during heme degradation by the enzyme heme oxygenase (HO) which is the third step of ring opening of verdoheme to biliverdin, a process maintaining iron homeostasis. The calculations highlighted the significant influence of the distal water cluster which acts as a biocatalyst for the conversion of verdoheme to biliverdin in the two processes, by fixing the departing OH and directing it to the requisite site of attack and by acting as a proton shuttle for the highly reactive OH(-) nucleophile.

The continuum electrostatics program multiconformation continuum (MCCE) is used to compare the electrochemical behavior of heme a in wild-type and S44D cytochrome c oxidase (CcO). The findings provide insight into the pH dependent stabilization of a heme oxidation in a model complex by a hydrogen bond to the axial His ligand.

The reduced state of the protein for which the B-state is prepared by ligating the ferrous heme iron with extrinsic CO under 1 mM gas concentration in the presence of NaCl was used to study the relevance of the alkali molten globule (B-state) to the folding pathway of cytochrome c. The alkali molten globule was not found to correspond to any possible transient structure in the folding pathway of cytochrome c.