H2O2-dependent substrate oxidation by an engineered diiron site in a bacterial hemerythrin.
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ABSTRACT:
SUBMITTER: Okamoto Y
PROVIDER: S-EPMC3947700 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ? 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.
| S-EPMC3873334 | biostudies-literature
Project description:The regulatory component (MMOB) of soluble methane monooxygenase (sMMO) has a unique N-terminal tail not found in regulatory proteins of other bacterial multicomponent monooxygenases. This N-terminal tail is indispensable for proper function, yet its solution structure and role in catalysis remain elusive. Here, by using double electron-electron resonance (DEER) spectroscopy, we show that the oxidation state of the hydroxylase component, MMOH, modulates the conformation of the N-terminal tail in the MMOH-2MMOB complex, which in turn facilitates catalysis. The results reveal that the N-terminal tail switches from a relaxed, flexible conformational state to an ordered state upon MMOH reduction from the diiron(III) to the diiron(II) state. This observation suggests that some of the crystallographically observed allosteric effects that result in the connection of substrate ingress cavities in the MMOH-2MMOB complex may not occur in solution in the diiron(III) state. Thus, O2 may not have easy access to the active site until after reduction of the diiron center. The observed conformational change is also consistent with a higher binding affinity of MMOB to MMOH in the diiron(II) state, which may allow MMOB to displace more readily the reductase component (MMOR) from MMOH following reduction.
| S-EPMC3954536 | biostudies-literature
Project description:Hydrogen peroxide (H2O2) is one of a variety of reactive oxygen species (ROS) produced by aerobic organisms. Host production of toxic H2O2 in response to pathogen infection is an important classical innate defense mechanism against invading microbes. Understanding the mechanisms by which pathogens, in response to oxidative stress, mediate defense against toxic ROS, can reveal anti-microbial targets and shed light on pathogenic mechanisms. In this study, we provide evidence that a Mycobacterium smegmatis hemerythrin-like protein MSMEG_2415, designated MsmHr, is a H2O2-modulated repressor of the SigF-mediated response to H2O2. Circular dichroism and spectrophotometric analysis of MsmHr revealed properties characteristic of a typical hemerythrin-like protein. An msmHr knockout strain of M. smegmatis mc(2)155 (?msmHr) was more resistant to H2O2 than its parental strain, and overexpression of MsmHr increased mycobacterial susceptibility to H2O2. Mutagenesis studies revealed that the hemerythrin domain of MsmHr is required for the regulation of the H2O2 response observed in the overexpression study. We show that MsmHr inhibits the expression of SigF (MSMEG_1804), an alternative sigma factor that plays an important role in bacterial oxidative stress responses, including those elicited by H2O2, thus providing a mechanistic link between ?msmHr and its enhanced resistance to H2O2. Together, these results strongly suggest that MsmHr is involved in the response of mycobacteria to H2O2 by negatively regulating a sigma factor, a function not previously described for hemerythrins.
| S-EPMC4295536 | biostudies-literature
Project description:The reaction of the ferrous complex [LFe(NCMe)2 ](OTf)2 (1), which contains a macrocyclic tetracarbene as ligand (L), with Na(OCP) generates the OCP- -ligated complex [LFe(PCO)(CO)]OTf (2) together with the dinuclear μ-phosphido complex [(LFe)2 P](OTf)3 (3), which features an unprecedented linear Fe-(μ-P)-Fe motif and a "naked" P-atom bridge that appears at δ=+1480 ppm in the 31 P NMR spectrum. 3 exhibits rich redox chemistry, and both the singly and doubly oxidized species 4 and 5 could be isolated and fully characterized. X-ray crystallography, spectroscopic studies, in combination with DFT computations provide a comprehensive electronic structure description and show that the Fe-(μ-P)-Fe core is highly covalent and structurally invariant over the series of oxidation states that are formally described as ranging from FeIII FeIII to FeIV FeIV . 3-5 now add a higher homologue set of complexes to the many systems with Fe-(μ-O)-Fe and Fe-(μ-N)-Fe core structures that are prominent in bioinorganic chemistry and catalysis.
| S-EPMC6790664 | biostudies-literature
Project description:Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O2. However, only FDP competently turns over NO to N2O. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (Hex = JS1·S2) was found to increase from +20 cm-1 to +32 cm-1 upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-?-hydroxo diferrous species is the catalytically functional state that reacts with NO and O2. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO.
| S-EPMC5898632 | biostudies-literature
Project description:Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.
| S-EPMC3972754 | biostudies-literature
Project description:Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV-vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)-tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)-tyrosyl radical center entails the formation of significant Cu(II)-(●OTyr) overlap, which in turn requires that the plane of the d(x2-y2) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a "hole-hopping" charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.
| S-EPMC7007232 | biostudies-literature
Project description:The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganese-dependent RNA ribose 2'O-methyltransferase that marks the 3' terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 3' dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12-24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 3' terminal ribonucleotide, signifying that it has no strict RNA specificity beyond the 3' nucleoside. CthHen1 methylated RNA ends in the context of duplex secondary structures. These properties distinguish bacterial Hen1 from plant and metazoan homologs.
| S-EPMC3039143 | biostudies-literature
Project description:The iron(II) triflate complex (1) of 1,2-bis(2,2'-bipyridyl-6-yl)ethane, with two bipyridine moieties connected by an ethane bridge, was prepared. Addition of aqueous 30% H2O2 to an acetonitrile solution of 1 yielded 2, a green compound with ?max=710 nm. Moessbauer measurements on 2 showed a doublet with an isomer shift (?) of 0.35 mm/s and a quadrupole splitting (?EQ) of 0.86 mm/s, indicative of an antiferromagnetically coupled diferric complex. Resonance Raman spectra showed peaks at 883, 556 and 451 cm-1 that downshifted to 832, 540 and 441 cm-1 when 1 was treated with H218O2. All the spectroscopic data support the initial formation of a (?-hydroxo)(?-1,2-peroxo)diiron(III) complex that oxidizes carbon-hydrogen bonds. At 0°C 2 reacted with cyclohexene to yield allylic oxidation products but not epoxide. Weak benzylic C-H bonds of alkylarenes were also oxidized. A plot of the logarithms of the second order rate constants versus the bond dissociation energies of the cleaved C-H bond showed an excellent linear correlation. Along with the observation that oxidation of the probe substrate 2,2-dimethyl-1-phenylpropan-1-ol yielded the corresponding ketone but no benzaldehyde, and the kinetic isotope effect, kH/kD , of 2.8 found for the oxidation of xanthene, the results support the hypothesis for a metal-based H-atom abstraction mechanism. Complex 2 is a rare example of a (?-hydroxo)(?-1,2-peroxo)diiron(III) complex that can elicit the oxidation of carbon-hydrogen bonds.
| S-EPMC5765857 | biostudies-literature
Project description:H(2)O(2) acts as a signaling molecule by oxidizing critical thiol groups on redox-regulated target proteins. To explain the efficiency and selectivity of H(2)O(2)-based signaling, it has been proposed that oxidation of target proteins may be facilitated by H(2)O(2)-scavenging peroxidases. Recently, a peroxidase-based protein oxidation relay has been identified in yeast, namely the oxidation of the transcription factor Yap1 by the peroxidase Orp1. It has remained unclear whether the protein oxidase function of Orp1 is a singular adaptation or whether it may represent a more general principle. Here we show that Orp1 is in fact not restricted to oxidizing Yap1 but can also form a highly efficient redox relay with the oxidant target protein roGFP (redox-sensitive green fluorescent protein) in mammalian cells. Orp1 mediates near quantitative oxidation of roGFP2 by H(2)O(2), and the Orp1-roGFP2 redox relay effectively converts physiological H(2)O(2) signals into measurable fluorescent signals in living cells. Furthermore, the oxidant relay phenomenon is not restricted to Orp1 as the mammalian peroxidase Gpx4 also mediates oxidation of proximal roGFP2 in living cells. Together, these findings support the concept that certain peroxidases harbor an intrinsic and powerful capacity to act as H(2)O(2)-dependent protein thiol oxidases when they are recruited into proximity of oxidizable target proteins.
| S-EPMC2797222 | biostudies-literature