Biomedical subjects

Radu Silaghi-Dumitrescu

Publications and source records attributed to Radu Silaghi-Dumitrescu.

15 recordsLinked to original sources

PubMed2007

Ferryl haem protonation gates peroxidatic reactivity in globins.

Ferryl (Fe(IV)=O) species are involved in key enzymatic processes with direct biomedical relevance; among others, the uncontrolled reactivities of ferryl Mb (myoglobin) and Hb (haemoglobin) have been reported to be central to the pathology of rhabdomyolysis and subarachnoid haemorrhage. Rapid-scan stopped-flow methods have been used to monitor the spectra of the ferryl species in Mb and Hb as a function of pH. The ferryl forms of both proteins display an optical transition with pK approximately 4.7, and this is assigned to protonation of the ferryl species itself. We also demonstrate for the first time a direct correlation between Hb/Mb ferryl reactivity and ferryl protonation status, simultaneously informing on chemical mechanism and toxicity and with broader biochemical implications.

Animals↗

PubMed2006

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo.

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.

Animals↗

PubMed2006

Structural basis for O2 sensing by the hemerythrin-like domain of a bacterial chemotaxis protein: substrate tunnel and fluxional N terminus.

The methyl-accepting chemotaxis protein, DcrH, from the anaerobic sulfate-reducing bacterium, Desulfovibrio vulgaris (Hildenborough), has a hemerythrin-like domain, DcrH-Hr, at its C terminus. DcrH-Hr was previously shown to contain a diiron site that binds O2, suggesting an O2-sensing function. X-ray crystal structures of diferric (met-), azido-diferric (azidomet-), and diferrous (deoxy-) DcrH-Hr reveal a "substrate tunnel" distinct from that in invertebrate hemerythrins. This tunnel is proposed to facilitate the rapid autoxidation of oxy-DcrH-Hr and suggests that sensing is triggered by O2 binding and subsequent oxidation of the diferrous active site. The N-terminal loop of DcrH-Hr is highly ordered in both met- and azidomet-DcrH-Hr but is disordered in deoxy-DcrH-Hr. These redox-dependent conformational differences presumably transduce the sensory signal of DcrH-Hr to the neighboring methylation domain in the full-length receptor. Given the putative cytoplasmic localization of its Hr-like O2-sensing domain, DcrH is proposed to serve a role in negative aerotaxis (anaerotaxis).

Bacterial Proteins↗

PubMed2006

Copper-containing nitrite reductase: a DFT study of nitrite and nitric oxide adducts.

Copper-containing nitrite reductases (Cu-NIRs) reduce nitrite to NO. Reported here are DFT (density functional theory) results on models of the Cu-NIR active site bound to nitrite and nitric oxide. The Cu-NIR active site appears to have been designed to exclude N-nitrite binding even though N-O bond cleavage would be equally facile in the N- and O-isomers. The active site also appears to force a side-on coordination of the end-product, nitric oxide. The latter feature has to rely on the sterics of the active site to destabilize, thermodynamically speaking, the Cu-NO adduct; under these conditions, the absence of N-nitrite coordination is proposed to be merely a side-effect. For the Cu(II)-NO adduct, sterical crowding appears to also favour the Cu-NO electromer over Cu(I)-NO+, helping to avoid the potentially damaging chemistry associated with an NO+ moiety. These conclusions are in reasonable agreement with previous conclusions drawn from experiment [Science 304 (2004) 867].

Catalytic Domain↗

PubMed2005

DFT and the electromerism in complexes of iron with diatomic ligands.

A reliable procedure is proposed for assigning the electronic structures for large biologically-relevant systems, where the size of the model confines one to the use of density functional theory (DFT) methods, and where the risk of over-interpreting DFT-derived molecular orbitals and spin densities still exists. The proposed approach focuses on the use of the only DFT-derived parameter that is unanimously recognized to be reliable: the geometry. We examine DFT-derived O-O bond lengths in formally ferrous-dioxygen models, and compare them to bond lengths in free, non metal-bound, dioxygen, superoxide and peroxide moieties. Likewise, we compare the N-O bond lengths within ferrous-nitrosyl {FeNO}7 models, with the same parameter in free NO+, NO*, and HNO species. This allows a calibrated, straightforward way of assigning the electronic structure in systems where electromerism makes detailed single-reference molecular orbital analysis unreliable.

Ferric Compounds↗

PubMed2005

Transient species involved in catalytic dioxygen/peroxide activation by hemoproteins: possible involvement of protonated Compound I species.

Interaction of hemoproteins with peroxide leads in several cases to transient formation of ferric peroxo, ferric hydroperoxo, and "high-valent", formally Fev, oxo or hydroxo Compound species. Here, density functional calculations on ferric peroxo, ferric hydroperoxo, Compound and protonated Compound heme active site models are reported. The theoretical results, including calculated isotropic Fermi contact couplings and anisotropic spin dipole couplings, are found to generally correlate well with experimental EPR/ENDOR data. Hydrogen bonding and solvation affect the ferric-peroxo/ferrous-superoxo electromerism. The transition between the two electromers appears smooth, but neither hydrogen bonding to up to two water molecules, nor solvation appear able to dramatically alter the redox state of the superoxo ligand or of the iron. The presence of almost one full unpaired electron on the iron and of one full unpaired electron on the dioxygenic ligand in the "ferric-peroxo" state suggests a possible description of non-protonated "ferric-peroxo" as {ferric-superoxo+porphyrin radical}. Emerging from the DFT data is the possibility that a protonated Compound has already been detected in ENDOR experiments on cytochrome P450. The general feasibility of a protonated Compound in P450 monooxygenases is probed in light of these findings. To encompass the multiple mechanisms available in P450 for substrate oxidation, we define "mechanistic promiscuity" as the feature allowing an enzyme to perform the same reaction, with the same product, using more than one mechanism.

Anisotropy↗

PubMed2005

High-resolution crystal structures of Desulfovibrio vulgaris (Hildenborough) nigerythrin: facile, redox-dependent iron movement, domain interface variability, and peroxidase activity in the rubrerythrins.

High-resolution crystal structures of Desulfovibrio vulgaris nigerythrin (DvNgr), a member of the rubrerythrin (Rbr) family, demonstrate an approximately 2-A movement of one iron (Fe1) of the diiron site from a carboxylate to a histidine ligand upon conversion of the mixed-valent ([Fe2(II),Fe1(III)]) to diferrous states, even at cryogenic temperatures. This Glu<-->His ligand "toggling" of one iron, which also occurs in DvRbr, thus, appears to be a characteristic feature of Rbr-type diiron sites. Unique features of DvNgr revealed by these structures include redox-induced flipping of a peptide carbonyl that reversibly forms a hydrogen bond to the histidine ligand to Fe1 of the diiron site, an intra-subunit proximal orientation of the rubredoxin-(Rub)-like and diiron domains, and an electron transfer pathway consisting of six covalent and two hydrogen bonds connecting the Rub-like iron with Fe2 of the diiron site. This pathway can account for DvNgr's relatively rapid peroxidase turnover. The characteristic combination of iron sites together with the redox-dependent iron toggling between protein ligands can account for the selectivity of Rbrs for hydrogen peroxide over dioxygen.

Bacterial Proteins↗

PubMed2005

X-ray crystal structures of Moorella thermoacetica FprA. Novel diiron site structure and mechanistic insights into a scavenging nitric oxide reductase.

Several members of a widespread class of bacterial and archaeal metalloflavoproteins, called FprA, likely function as scavenging nitric oxide reductases (S-NORs). However, the only published X-ray crystal structure of an FprA is for a protein characterized as a rubredoxin:dioxygen oxidoreductase (ROO) from Desulfovibrio gigas. Therefore, the crystal structure of Moorella thermoacetica FprA, which has been established to function as an S-NOR, was solved in three different states: as isolated, reduced, and reduced, NO-reacted. As is the case for D. gigas ROO, the M. thermoacetica FprA contains a solvent-bridged non-heme, non-sulfur diiron site with five-coordinate iron centers bridged by an aspartate, and terminal glutamate, aspartate, and histidine ligands. However, the M. thermoacetica FprA diiron site showed four His ligands, two to each iron, in all three states, whereas the D. gigas ROO diiron site was reported to contain only three His ligands, even though the fourth His residue is conserved. The Fe1-Fe2 distance within the diiron site of M. thermoacetica FprA remained at 3.2-3.4 A with little or no movement of the protein ligands in the three different states and with conservation of the two proximal open coordination sites. Molecular modeling indicated that each open coordination site can accommodate an end-on NO. This relatively rigid and symmetrical diiron site structure is consistent with formation of a diferrous dinitrosyl as the committed catalytic intermediate leading to formation of N(2)O. These results provide new insight into the structural features that fine-tune biological non-heme diiron sites for dioxygen activation vs nitric oxide reduction.

Binding Sites↗

PubMed2005

A flavo-diiron protein from Desulfovibrio vulgaris with oxidase and nitric oxide reductase activities. Evidence for an in vivo nitric oxide scavenging function.

A few members of a widespread class of bacterial and archaeal flavo-diiron proteins, dubbed FprAs, have been shown to function as either oxidases (dioxygen reductases) or scavenging nitric oxide reductases, but the questions of which of these functions dominates in vivo for a given FprA and whether all FprAs function as oxidases or nitric oxide reductases remain to be clarified. To address these questions, an FprA has been characterized from the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris. The gene encoding this D. vulgaris FprA lies downstream of an operon encoding superoxide reductase and rubredoxin, consistent with an O(2)-scavenging oxidase function for this FprA. The recombinant D. vulgaris FprA can indeed serve as the terminal component of an NADH oxidase. However, this oxidase turnover results in irreversible inactivation of the enzyme. On the other hand, the recombinant D. vulgaris FprA shows robust anaerobic nitric oxide reductase activity in vitro and also protects a nitric oxide-sensitive Escherichia coli strain against exposure to exogenous nitric oxide. It is, therefore, proposed that this D. vulgaris FprA functions as a scavenging nitric oxide reductase in vivo and that this activity protects D. vulgaris against anaerobic exposure to nitric oxide. The location of a gene encoding a second FprA homologue in the D. vulgaris genome also suggests its involvement in nitrogen oxide metabolism.

Bacterial Proteins↗

PubMed2005

Cytochrome bd oxidase, oxidative stress, and dioxygen tolerance of the strictly anaerobic bacterium Moorella thermoacetica.

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (approximately 70%) inhibited both the endogenous and CO/methanol-dependent O2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-beta-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b561, cytochrome b595, and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine + H2S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.

Amino Acid Sequence↗

PubMed2004

Linkage isomerism in nitrite reduction by cytochrome cd1 nitrite reductase.

Nitrite reduction by cytochrome cd(1) nitrite reductase (cd(1)NIR) is currently accepted to involve coordination of the nitrite nitrogen atom to the ferrous d(1) heme. Here, density functional theory results are reported on the previously unexplored O-binding of nitrite to ferrous and ferric cd(1)NIR. Although the N-isomer (nitro) is energetically favored over the O-nitrite (nitrito), even one single strong hydrogen bond may provide the energy required to put the two isomers on level terms. When hydrogen bonding existent at the cd(1)NIR active site is accounted for in the computational model, the O-nitrite isomer is found to spontaneously protonate and thus yield a ferric-hydroxo species, liberating nitric oxide. An O-nitrite ferrous cd(1)NIR complex appears to be an energetically feasible intermediate for nitrite reduction. O-Coordination would offer an advantage since the end product of nitrite reduction would be a ferric-hydroxo/water complex, rather than the more kinetically inert iron-nitrosyl complex implied by the N-nitrite mechanism.

Cytochromes↗

PubMed2004

The nature of the high-valent complexes in the catalytic cycles of hemoproteins.

We report geometry optimization results on heme compound I (ferryl-oxo + porphyrin cation radical), compound II (ferryl-oxo) and ferric-hydroxo species with thiolate or imidazole axial ligands. We also examine protonated forms of compound I and compound II species, prompted by recent reports that, in at least two different hemoproteins, compound II may in fact contain a hydroxo rather than an oxo ligand. We propose that the stable compound I and compound II species of hemoproteins (e.g., peroxidases and myoglobin) most likely contain a hydroxo rather than the oxo ligand traditionally assumed, whereas the extremely transient compound I species of monooxygenase hemoproteins (P450) would contain an oxo atom. We show evidence impacting the previously accepted notion in hemoprotein computational chemistry that non-covalent interactions and medium polarization effects are essential in properly describing the electronic structure of heme-thiolate high-valent complexes. On a different note, we find that the charge density on the iron remains essentially the same throughout the catalytic cycles of heme-containing oxygenases and peroxidases, despite clear changes in bond lengths and spin densities suggestive of various iron oxidation states. The iron thus appears to simply relay the electron flux between the porphyrin and the axial dioxygen/superoxo/peroxo/oxo/hydroxo ligands.

Catalysis↗

PubMed2004

Heme ferrous-hydroperoxo complexes: some theoretical considerations.

We report density functional calculations on complexes of ferrous hemes with hydroperoxide, where the axial ligand trans to OOH(-) is imidazole, thiolate, or phenoxide. The geometrical parameters and charge distributions within the Fe-O-O-H moiety are identical between the ferrous complexes reported here and their ferric counterparts previously described, even though the latter contain one unpaired electron on iron as opposed to the former, which are diamagnetic. The extra negative charge upon going from a formally ferric state to formally ferrous appears to be distributed essentially on the porphyrin. These findings support recent experimental data showing that the ferrous state of certain hemoproteins can interact with peroxides in a catalytically competent fashion, cleaving the O-O bond heterolytically in a manner reminiscent of the "canonical" ferric-peroxo complexes, and contrary to any expectations based on the Fenton concept commonly invoked in non-heme chemistry.

Computer Simulation↗

PubMed2003

A flavodiiron protein and high molecular weight rubredoxin from Moorella thermoacetica with nitric oxide reductase activity.

A five-gene "oxidative stress protection" cluster has recently been described from the strictly anaerobic, acetogenic bacterium, Moorella thermoacetica [Das, A., et al. (2001) J. Bacteriol. 183, 1560-1567]. Within this cluster are two cotranscribed genes, fprA (for A-type flavoprotein) and hrb (for high molecular weight rubredoxin) whose encoded proteins have no known functions. Here we show that FprA and Hrb are expressed in M. thermoacetica under normal anaerobic growth conditions and report characterizations of the recombinant FprA and Hrb. FprA contains flavin mononucleotide (FMN) and a non-heme diiron site. Mössbauer spectroscopy shows that the irons of the diferric site are antiferromagnetically coupled, implying a single-atom, presumably solvent, bridge between the irons. Hrb contains FMN and a rubredoxin-like [Fe(SCys)4] site. NADH does not directly reduce either the FMN or the diiron site in FprA, whereas Hrb functions as an efficient NADH:FprA oxidoreductase. Substitution of zinc for iron in Hrb completely abolished this activity. The observation that homologues of FprA from other organisms show O2 and/or anaerobic NO consumption activity prompted an examination of these activities for M. thermoacetica FprA. The Hrb/FprA combination does indeed have both NADH:O2 and NADH:NO oxidoreductase activities. The NO reductase activity, however, was significantly more efficient due to a lower Km for NO (4 M) and to progressive and irreversible inactivation of FprA during O2 reductase turnover but retention of activity during NO reductase turnover. Substitution of zinc for iron in FprA completely abolished these reductase activities. The stoichiometry of 1 mol of NADH oxidized:2 mol of NO consumed implies reduction to N2O. Fits of an appropriate rate law to the kinetics data are consistent with a mechanism in which 2NO's react at each FprA active site in the committed step. Expression of FprA in an Escherichia coli strain deficient in NO reductase restored the anaerobic growth phenotype of cultures exposed to otherwise toxic levels of exogenous NO. The accumulated results indicate that Hrb/FprA is fully capable of functioning in nitrosative stress protection in M. thermoacetica.

Bacterial Proteins↗

PubMed2003

Computational study of the non-heme iron active site in superoxide reductase and its reaction with superoxide.

The ferrous square-pyramidal [Fe(NHis)4(SCys)] site of superoxide reductases (SORs) has been shown to reduce superoxide at a nearly diffusion-controlled rate. The final products of the reaction are hydrogen peroxide and the ferric hexacoordinated SOR site, with a carboxylate group from a conserved glutamate serving as the sixth ligand trans to the cysteine sulfur. A transient intermediate absorbing at approximately 600 nm in the reaction of the ferrous pentacoordinated site with superoxide has been proposed to be a ferric-(hydro)peroxo complex (Coulter, E.; Emerson, J.; Kurtz, D. M., Jr.; Cabelli, D. J. Am. Chem. Soc. 2000, 122, 11555-11556.). In the present study, DFT and ZINDO/S-CI results are shown to support the description of the 600-nm intermediate as an end-on, low-spin ferricperoxo or--hydroperoxo complex. Side-on peroxo coordination was found to be significantly less stable than end-on because of constraints on the imidazole ligand ring orientations imposed mostly by the protein. The modeled ferric-hydroperoxo complex had a decidedly nonplanar CysC beta-S-Fe-O-O geometry that appears to be imposed by the same constraints. A single prominent visible absorption of the (hydro)peroxo model is shown to be due mainly to a CysS-->Fe(III) pi charge transfer (CT) transition with a minor portion of His-->Fe(III) pi CT character and very little peroxo-->Fe(III) CT character. On the basis of calculations of models with various mono- and diprotonated peroxo ligands, protonation of the iron-bound peroxo oxygen is a key step in the decay of the ferric(hydro)peroxo complex favoring release of hydrogen peroxide over cleavage of the O-O bond, as occurs in the heme structural analogue, cytochrome P450.

Binding Sites↗