The critical role of a conserved lysine residue in periplasmic nitrate reductase catalyzed reactions.

Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.

Oxidative Dearomatization of PLP in Thiamin Pyrimidine Biosynthesis in Candida albicans.

The yeast thiamin pyrimidine synthase THI5p catalyzes one of the most complex organic rearrangements found in primary metabolism. In this reaction, the active site His66 and PLP are converted to thiamin pyrimidine in the presence of Fe(II) and oxygen. The enzyme is a single-turnover enzyme. Here, we report the identification of an oxidatively dearomatized PLP intermediate. We utilize oxygen labeling studies, chemical-rescue-based partial reconstitution experiments, and chemical model studies to support this identification. In addition, we also identify and characterize three shunt products derived from the oxidatively dearomatized PLP.

Trapping and structural characterisation of a covalent intermediate in vitamin B6 biosynthesis catalysed by the Pdx1 PLP synthase.

The Pdx1 enzyme catalyses condensation of two carbohydrates and ammonia to form pyridoxal 5-phosphate (PLP) via an imine relay mechanism of carbonyl intermediates. The I333 intermediate characterised here using structural, UV-vis absorption spectroscopy and mass spectrometry analyses rationalises stereoselective deprotonation and subsequent substrate assisted phosphate elimination, central to PLP biosynthesis.

Optimal control of photodissociation of phenol using genetic algorithm.

Photodissociation dynamics of the OH bond of phenol is studied with an optimally shaped laser pulse. The theoretical model consists of three electronic states (the ground electronic state, ππ* state, and πσ* state) in two nuclear coordinates (the OH stretching coordinate as a reaction coordinate, r, and the CCOH dihedral angle as a coupling coordinate, θ). The optimal UV laser pulse is designed using the genetic algorithm, which optimizes the total dissociative flux of the wave packet. The latter is calculated in the adiabatic asymptotes of the S0 and S1 electronic states of phenol. The initial state corresponds to the vibrational levels of the electronic ground state and is defined as |nr, nθ⟩, where nr and nθ represent the number of nodes along r and θ, respectively. The optimal UV field excites the system to the optically dark πσ* state predominantly over the optically bright ππ* state with the intensity borrowing effect for the |0, 0⟩ and |0, 1⟩ initial states. For the |0, 0⟩ initial condition, the photodissociation to the S1 asymptotic channel is favored slightly over the S0 asymptotic channel. Addition of one quantum of energy along the coupling coordinate increases the dissociation probability in the S1 channel. This is because the wave packet spreads along the coupling coordinate on the πσ* state and follows the adiabatic path. Hence, the S1 asymptotic channel gets more (∼11%) dissociative flux as compared to the S0 asymptotic channel for the |0, 1⟩ initial condition. The |1, 0⟩ and |1, 1⟩ states are initially excited to both the ππ* and πσ* states in the presence of the optimal UV pulse. For these initial conditions, the S1 channel gets more dissociative flux as compared to the S0 channel. This is because the high energy components of the wave packet readily reach the S1 channel. The central frequency of the optimal UV pulse for the |0, 0⟩ and |0, 1⟩ initial states has a higher value as compared to the |1, 0⟩ and |1, 1⟩ initial states. This is explained with the help of an excitation mechanism of a given initial state in relation to its energy.

Spectroscopy and DFT Calculations of Flavo-Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates.

Flavo-diiron proteins (FDPs) are widespread in anaerobic bacteria, archaea, and protozoa, where they serve as the terminal components of dioxygen and nitric oxide reductive scavenging pathways. FDPs contain an N,O-ligated diiron site adjacent to a flavin mononucleotide (FMN) cofactor. The diiron site is structurally similar to those in hemerythrin, ribonucleotide reductase, and methane monooxygenase. However, only FDPs turn over NO to N2O at significant rates and yields. Previous studies revealed sequential binding of two NO molecules to the diferrous site, forming mono- and dinitrosyl intermediates leading to N2O formation. In the present work, these mono- and dinitrosyl intermediates have been characterized by EPR and Mössbauer spectroscopies and DFT calculations. Our results show that the iron proximal to the cofactor binds the first NO to form the diiron mononitrosyl complex, implying the iron distal to the FMN binds the second NO to form the diiron dinitrosyl intermediate. The exchange-coupling constants, J (H = JS1·S2), were found to differ substantially, +17 cm-1 for the diiron mononitrosyl and +60 cm-1 for the diiron dinitrosyl. Notwithstanding this large difference, our findings indicate retention of at least one hydroxo bridge throughout the NOR catalytic cycle. The Mossbauer hyperfine parameters and DFT calculations confirmed a semibridging NO- ligand in the mononitrosyl intermediate that lowers the exchange parameter. The DFT calculations on the dinitrosyl intermediate suggest a contribution to J from direct exchange between the S = 1 spins on the NO- ligands, which could initiate N-N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N2O.

Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures.

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-µ-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.

Influence of microwaves on the leaching kinetics of uraninite from a low grade ore in dilute sulfuric acid.

This paper describes a study on microwave assisted leaching of uranium from a low-grade ore of Indian origin. The host rock for uranium mineralization is chlorite-biotite-muscovite-quartzo-feldspathic schist. The dominant presence of siliceous minerals determined leaching of uranium values in sulfuric acid medium under oxidizing conditions. Process parametric studies like the effect of sulfuric acid concentration (0.12-0.50M), redox potential (400-500mV), particle size (600-300µm) and temperature (35°-95°C) indicated that microwave assisted leaching is more efficient in terms of overall uranium dissolution, kinetics and provide relatively less impurities (Si, Al, Mg and Fe) in the leach liquor compared to conventional conductive leaching. The kinetics of leaching followed shrinking core model with product layer diffusion as controlling mechanism.

A diferrous-dinitrosyl intermediate in the N2O-generating pathway of a deflavinated flavo-diiron protein.

Flavo-diiron proteins (FDPs) function as anaerobic nitric oxide scavengers in some microorganisms, catalyzing reduction of nitric to nitrous oxide. The FDP from Thermotoga maritima can be prepared in a deflavinated form with an intact diferric site (deflavo-FDP). Hayashi et al. [(2010) Biochemistry 49, 7040-7049] reported that reaction of NO with reduced deflavo-FDP produced substoichiometric N2O. Here we report a multispectroscopic approach to identify the iron species in the reactions of deflavo-FDP with NO. Mössbauer spectroscopy identified two distinct ferrous species after reduction of the antiferromagnetically coupled diferric site. Approximately 60% of the total ferrous iron was assigned to a diferrous species associated with the N2O-generating pathway. This pathway proceeds through successive diferrous-mononitrosyl (S = (1)/2 Fe(II){FeNO}(7)) and diferrous-dinitrosyl (S = 0 [{FeNO}(7)]2) species that form within ∼100 ms of mixing of the reduced protein with NO. The diferrous-dinitrosyl intermediate converted to an antiferromagnetically coupled diferric species that was spectroscopically indistinguishable from that in the starting deflavinated protein. These diiron species closely resembled those reported for the flavinated FDP [Caranto et al. (2014) J. Am. Chem. Soc. 136, 7981-7992], and the time scales of their formation and decay were consistent with the steady state turnover of the flavinated protein. The remaining ∼40% of ferrous iron was inactive in N2O generation but reversibly bound NO to give an S = (3)/2 {FeNO}(7) species. The results demonstrate that N2O formation in FDPs can occur via conversion of S = 0 [{FeNO}(7)]2 to a diferric form without participation of the flavin cofactor.

Structural investigations of the nickel-induced inhibition of truncated constructs of the JMJD2 family of histone demethylases using X-ray absorption spectroscopy.

Occupational and/or environmental exposure to nickel has been implicated in various types of cancer, and in vitro exposure to nickel compounds results in the accumulation of Ni(II) ions in cells. One group of major targets of Ni(II) ions inside the cell consists of Fe(II)- and αKG-dependent dioxygenases. Using JMJD2A and JMJD2C as examples, we show that the JMJD2 family of histone demethylases, which are products of putative oncogenes as well as Fe(II)- and αKG-dependent dioxygenases, are highly sensitive to inhibition by Ni(II) ions. In this work, X-ray absorption spectroscopy (XAS) has been used to investigate the Fe(II) active site of truncated JMJD2A and JMJD2C (1-350 amino acids) in the presence and absence of αKG and/or substrate to obtain mechanistic details of the early steps in catalysis that precede O2 binding in histone demethylation by the JMJD2 family of histone demethylases. Zinc K-edge XAS has been performed on the resting JMJD2A (with iron in the active site) to confirm the presence of the expected structural zinc site. XAS of the Ni(II)-substituted enzymes has also been performed to investigate the inhibition of these enzymes by Ni(II) ions. Our XAS results indicate that the five-coordinate Fe(II) center in the resting enzyme is retained in the binary and ternary complexes. In contrast, the Ni(II) center is six-coordinate in the resting enzyme and binary and ternary complexes. XAS results indicate that both Fe(II) and Ni(II) bind αKG in the binary and ternary complexes. The electron density buildup that is observed at the Fe(II) center in the presence of αKG and substrate is not observed at the Ni(II) center. Thus, both electronic and steric factors are responsible for Ni-induced inhibition of the JMJD2 family of histone demethylases. Ni-induced inhibition of these enzymes may explain the alteration of the epigenetic mechanism of gene expression that is responsible for Ni-induced carcinogenesis.

Inverse solvent isotope effects demonstrate slow aquo release from hypoxia inducible factor-prolyl hydroxylase (PHD2).

Prolyl hydroxylase domain 2 (PHD2) is deemed a primary oxygen sensor in humans, yet many details of its underlying mechanism are still not fully understood. (Fe(2+) + αKG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying three coordination sites, a bidentate α-ketoglutarate occupying two sites, and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O(2) to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with k(cat) exhibiting a kinetic pK(a) = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe(2+) + αKG)PHD2 at pH 6.50 with pH 8.50. The near-UV circular dichroism spectrum was virtually unchanged at elevated pH, indicating that the secondary structure did not change as a function of pH. UV-visible and Fe X-ray absorption spectroscopy indicated that the primary coordination sphere of Fe(2+) changed upon increasing the pH; extended X-ray absorption fine structure analysis found a short Fe-(O/N) bond length of 1.96 Å at pH 8.50, strongly suggesting that the aquo ligand was deprotonated at this pH. Solvent isotope effects were measured during steady-sate turnover over a wide pH-range, with an inverse solvent isotope effect (SIE) of k(cat) observed ((D(2)O)k(cat) = 0.91 ± 0.03) for the acid form; a similar SIE was observed for the basic form of the enzyme ((D(2)O)k(cat) = 0.9 ± 0.1), with an acid equilibrium offset of ΔpK(a) = 0.67 ± 0.04. The inverse SIE indicated that aquo release from the active site Fe(2+) immediately precedes a rate-limiting step, suggesting that turnover in this enzyme may be partially limited by the rate of O(2) binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pK(a) further suggested that PHD2 might function physiologically to sense both intracellular pO(2) as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2.

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.

Nickel ions inhibit histone demethylase JMJD1A and DNA repair enzyme ABH2 by replacing the ferrous iron in the catalytic centers.

Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC(50) [Ni(II)]) are 25 microm for JMJD1A and 7.5 microm for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC(50) [Ni(II)] of 2.5 microm, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mm concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development.