Addressing Ligand-Based Redox in Molybdenum-Dependent Methionine Sulfoxide Reductase.

A combination of pulsed EPR, CW EPR, and X-ray absorption spectroscopies has been employed to probe the geometric and electronic structure of the periplasmic molybdenum-dependent methionine sulfoxide reductase (MsrP). O and H pulsed EPR spectra show that the Mo(V) enzyme form does not possess an exchangeable HO/OH ligand bound to Mo as found in the sulfite oxidizing enzymes of the same family. The nature of the unusual CW EPR spectrum has been re-evaluated in light of new data on the MsrP-N45R variant and related small-molecule analogues of the active site.

The quinone-binding site of Acidithiobacillus ferrooxidans sulfide: quinone oxidoreductase controls both sulfide oxidation and quinone reduction.

Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane enzyme that catalyzes the oxidation of sulfide and the reduction of ubiquinone. Ubiquinone binds to a conserved hydrophobic domain and shuttles electrons from a noncovalent flavin adenine dinucleotide cofactor to the membrane-bound quinone pool. Utilizing the structure of decylubiquinone bound to Acidithiobacillus ferrooxidans SQR, we combined site-directed mutagenesis and kinetic approaches to analyze quinone binding.

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme.

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized "as-isolated" Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide.

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase.

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)).

Redox state of flavin adenine dinucleotide drives substrate binding and product release in Escherichia coli succinate dehydrogenase.

The Complex II family of enzymes, comprising respiratory succinate dehydrogenases and fumarate reductases, catalyzes reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, soluble fumarate reductases (e.g.

A simple semi-quantitative in vivo method using H₂S detection to monitor sulfide metabolizing enzymes.

Here we present a simple in vivo microtiter plate assay using lead acetate [Pb(OAc)2]-soaked filter paper to detect H2S released by Escherichia coli metabolizing cysteine. The released H2S precipitates as brown lead sulfide (PbS) on Pb(OAc)2 soaked filter paper. The PbS stain quantitated by ImageJ software is proportional to the amount of H2S released from the culture.

Characterization of the kinetics and electron paramagnetic resonance spectroscopic properties of Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR).

Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR) catalyzes the oxidation of sulfide to polysulfide chains or elemental sulfur coupled to quinone reduction via a non-covalent FAD cofactor. We investigated the role of the FAD using kinetics and EPR spectroscopy. The properties of the enzyme were compared with alanine and/or serine variants of conserved cysteine residues (Cys128, Cys160, Cys356) structurally close to the FAD cofactor and histidine residues (His132, His198) implicated in function.

Shifting the metallocentric molybdoenzyme paradigm: the importance of pyranopterin coordination.

In this review, we test the hypothesis that pyranopterin coordination plays a critical role in defining substrate reactivities in the four families of mononuclear molybdenum and tungsten enzymes (Mo/W-enzymes). Enzyme families containing a single pyranopterin dithiolene chelate have been demonstrated to have reactivity towards two (sulfite oxidase, SUOX-fold) and five (xanthine dehydrogenase, XDH-fold) types of substrate, whereas the major family of enzymes containing a bis-pyranopterin dithiolene chelate (dimethylsulfoxide reductase, DMSOR-fold) is reactive towards eight types of substrate. A second bis-pyranopterin enzyme (aldehyde oxidoreductase, AOR-fold) family catalyzes a single type of reaction.

A new paradigm for electron transfer through Escherichia coli nitrate reductase A.

We have investigated the role of redox cooperativity in defining the functional relationship among the three membrane-associated prosthetic groups of Escherichia coli nitrate reductase A: the two hemes (bD and bP) of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster (FS4) of the electron-transfer subunit (NarH). Previously published analyses of potentiometric titrations have exhibited the following anomalous behaviors: (i) fits of titration data for heme bp and the [3Fe-4S] cluster exhibited two apparent components; (ii) heme bD titrated with an apparent electron stoichiometry (n) of <1.0; and (iii) the binding of quinol oxidation inhibitors shifted the reduction potentials of both hemes despite there being only a single quinol oxidation site (Q-site) in close juxtaposition with heme bD.

Q-site occupancy defines heme heterogeneity in Escherichia coli nitrate reductase A (NarGHI).

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy.

Electron-transfer pathways in the heme and quinone-binding domain of complex II (succinate dehydrogenase).

Single electron transfers have been examined in complex II (succinate:ubiquinone oxidoreductase) by the method of pulse radiolysis. Electrons are introduced into the enzyme initially at the [3Fe-4S] and ubiquinone sites followed by intramolecular equilibration with the b heme of the enzyme. To define thermodynamic and other controlling parameters for the pathways of electron transfer in complex II, site-directed variants were constructed and analyzed.

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes.

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes.

Comparative proteomic and metabolomic analysis of Staphylococcus warneri SG1 cultured in the presence and absence of butanol.

The complete genome of the solvent tolerant Staphylococcus warneri SG1 was recently published. This Gram-positive bacterium is tolerant to a large spectrum of organic solvents including short-chain alcohols, alkanes, esters and cyclic aromatic compounds. In this study, we applied a two-dimensional liquid chromatography (2D-LC) mass spectrometry (MS) shotgun approach, in combination with quantitative 2-MEGA (dimethylation after guanidination) isotopic labeling, to compare the proteomes of SG1 grown under butanol-free and butanol-challenged conditions.

A conserved lysine residue controls iron-sulfur cluster redox chemistry in Escherichia coli fumarate reductase.

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair.

Complete Genome of the Solvent-Tolerant Staphylococcus warneri Strain SG1.

Staphylococcus warneri is a Gram-positive bacterium commonly found in human skin flora. The genome of a laboratory S. warneri isolate, strain SG1, was sequenced to explore its mechanism of solvent tolerance and its potential as a chassis for biofuel production.

A variant conferring cofactor-dependent assembly of Escherichia coli dimethylsulfoxide reductase.

We have investigated the final steps of complex iron-sulfur molybdoenzyme (CISM) maturation using Escherichia coli DMSO reductase (DmsABC) as a model system. The catalytic subunit of this enzyme, DmsA, contains an iron-sulfur cluster (FS0) and a molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD). We have identified a variant of DmsA (Cys59Ser) that renders enzyme maturation sensitive to molybdenum cofactor availability.

Pyranopterin conformation defines the function of molybdenum and tungsten enzymes.

We have analyzed the conformations of 319 pyranopterins in 102 protein structures of mononuclear molybdenum and tungsten enzymes. These span a continuum between geometries anticipated for quinonoid dihydro, tetrahydro, and dihydro oxidation states. We demonstrate that pyranopterin conformation is correlated with the protein folds defining the three major mononuclear molybdenum and tungsten enzyme families, and that binding-site micro-tuning controls pyranopterin oxidation state.

A protein export pathway involving Escherichia coli porins.

Escherichia coli export the protein YebF into the extracellular medium by a two-step process. However, as no general outer membrane protein secretion system common to all E. coli strains has been reported, the mechanism of export has remained unclear.

Structure-activity characterization of sulfide:quinone oxidoreductase variants.

Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane protein that catalyzes the oxidation of sulfide species to elemental sulfur. The enzymatic reaction proceeds in two steps. The electrons from sulfides are transferred first to the enzyme cofactor, FAD, which, in turn, passes them onto the quinone pool in the membrane.

Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase.

The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions.

Correct assembly of iron-sulfur cluster FS0 into Escherichia coli dimethyl sulfoxide reductase (DmsABC) is a prerequisite for molybdenum cofactor insertion.

The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = 3/2) in its reduced form, resulting in an EPR spectrum with a peak at g ∼ 5.

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster.

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster.

Molybdenum site structure of Escherichia coli YedY, a novel bacterial oxidoreductase.

We report a structural characterization using X-ray absorption spectroscopy of the molybdenum site of Escherichia coli YedY, a novel oxidoreductase related to be the sulfite oxidase family of molybdenum enzymes. We find that the enzyme can exist in Mo(V) and Mo(IV) oxidation states but cannot be readily oxidized to the Mo(VI) form. Mo(V) YedY has molybdenum coordination similar to that of sulfite oxidase, with one Mo═O at 1.

Crystal structure of sulfide:quinone oxidoreductase from Acidithiobacillus ferrooxidans: insights into sulfidotrophic respiration and detoxification.

Sulfide:quinone oxidoreductase from the acidophilic and chemolithotrophic bacterium Acidithiobacillus ferrooxidans was expressed in Escherichia coli and crystallized, and its X-ray molecular structure was determined to 2.3 A resolution for native unbound protein in space group P4(2)2(1)2 . The decylubiquinone-bound structure and the Cys160Ala variant structure were subsequently determined to 2.

Protein crystallography reveals a role for the FS0 cluster of Escherichia coli nitrate reductase A (NarGHI) in enzyme maturation.

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His(49) both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant.