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., those from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and FAD was examined. Variants SdhA-R286A/K/Y and -H242A/Y that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in the assembly of a noncovalent FAD cofactor, which led to a significant decrease (-87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The "free" and "occupied" states of the active site were linked to the reduced and oxidized states of FAD, respectively. Our data allow for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD.

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. The close proximity of these residues to the [3Fe-4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe-4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe-4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe-4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (>100mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.

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. In total, 1585 unique proteins (representing 65% of the predicted open reading frames) were identified, covering all major metabolic pathways. Of the 967 quantifiable proteins by 2-MEGA labeling, 260 were differentially expressed by at least 1.5-fold. These proteins are involved in energy metabolism, oxidative stress response, lipid and cell envelope biogenesis, or have chaperone functions. We also applied differential isotope labeling LC-MS to probe metabolite changes in key metabolic pathways upon butanol stress. This is the first comprehensive proteomic and metabolomic study of S. warneri SG1 and presents an important step toward understanding its physiology and mechanism of solvent tolerance.

Alternative sites for proton entry from the cytoplasm to the quinone binding site in Escherichia coli succinate dehydrogenase.

Escherichia coli succinate dehydrogenase (Sdh) belongs to the highly conserved complex II family of enzymes that reduce ubiquinone. These enzymes do not generate a protonmotive force during catalysis and are electroneutral. Because of its electroneutrality, the quinone reduction reaction must consume cytoplasmic protons which are released stoichiometrically during succinate oxidation. The X-ray crystal structure of E. coli Sdh shows that residues SdhB (G227), SdhC (D95), and SdhC (E101) are located at or near the entrance of a water channel that has been proposed to function as a proton wire connecting the cytoplasm to the quinone binding site. However, the pig and chicken Sdh enzymes show an alternative entrance to the water channel via the conserved SdhD (Q78) residue. In this study, site-directed mutants of these four residues were created and characterized by in vivo growth assays, in vitro activity assays, and electron paramagnetic resonance spectroscopy. We show that the observed water channel in the E. coli Sdh structure is the functional proton wire in vivo, while in vitro results indicate an alternative entrance for protons. In silico examination of the E. coli Sdh reveals a possible H-bonding network leading from the cytoplasm to the quinone binding site that involves SdhD (D15). On the basis of these results we propose an alternative proton pathway in E. coli Sdh that might be functional only in vitro.

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.

S- and N-Oxide Reductases.

Escherichia coli is a versatile facultative anaerobe that can respire on a number of terminal electron acceptors, including oxygen, fumarate, nitrate, and S- and N-oxides. Anaerobic respiration using S- and N-oxides is accomplished by enzymatic reduction of these substrates by dimethyl sulfoxide reductase (DmsABC) and trimethylamine N-oxide reductase (TorCA). Both DmsABC and TorCA are membrane-associated redox enzymes that couple the oxidation of menaquinol to the reduction of S- and N-oxides in the periplasm. DmsABC is membrane bound and is composed of a membrane-extrinsic dimer with a 90.4-kDa catalytic subunit (DmsA) and a 23.1-kDa electron transfer subunit (DmsB). These subunits face the periplasm and are held to the membrane by a 30.8-kDa membrane anchor subunit (DmsC). The enzyme provides the scaffold for an electron transfer relay composed of a quinol binding site, five [4Fe-4S] clusters, and a molybdo-bis(molybdopterin guanine dinucleotide) (present nomenclature: Mo-bis-pyranopterin) (Mo-bisMGD) cofactor. TorCA is composed of a soluble periplasmic subunit (TorA, 92.5 kDa) containing a Mo-bis-MGD. TorA is coupled to the quinone pool via a pentaheme c subunit (TorC, 40.4 kDa) in the membrane. Both DmsABC and TorCA require system-specific chaperones (DmsD or TorD) for assembly, cofactor insertion, and/or targeting to the Tat translocon. In this chapter, we discuss the complex regulation of the dmsABC and torCAD operons, the poorly understood paralogues, and what is known about the assembly and translocation to the periplasmic space by the Tat translocon.

The iron-sulfur clusters in Escherichia coli succinate dehydrogenase direct electron flow.

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.

Investigation of the environment surrounding iron-sulfur cluster 4 of Escherichia coli dimethylsulfoxide reductase.

Iron-sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E(m) values) play a major role in defining the rate at which electrons are shuttled. The E(m) values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1-FS4) with E(m) values between -50 and -330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH(2)) binding, and protein control of its E(m). Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB-DmsC interface and are critical for the binding of the MQH(2) inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH(2) to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin-spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4([3Fe)(-)(4S])). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E(m) of FS4([3Fe)(-)(4S]) from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4([3Fe)(-)(4S]) lower its E(m), but mutations of negatively charged residues have negligible effects. The E(m) of FS4([3Fe)(-)(4S]) in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4([3Fe)(-)(4S]) E(m) of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E(m) for FS4([3Fe)(-)(4S]) of DmsB is approximately 275 mV.