Defining a direction: electron transfer and catalysis in Escherichia coli complex II enzymes.
ABSTRACT: There are two homologous membrane-bound enzymes in Escherichia coli that catalyze reversible conversion between succinate/fumarate and quinone/quinol. Succinate:ubiquinone reductase (SQR) is a component of aerobic respiratory chains, whereas quinol:fumarate reductase (QFR) utilizes menaquinol to reduce fumarate in a final step of anaerobic respiration. Although, both protein complexes are capable of supporting bacterial growth on either minimal succinate or fumarate media, the enzymes are more proficient in their physiological directions. Here we evaluate factors that may underlie this catalytic bias. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.
Project description:The complex II family of proteins includes succinate:quinone oxidoreductase (SQR) and quinol:fumarate oxidoreductase (QFR). In the facultative bacterium Escherichia coli both are expressed as part of the aerobic (SQR) and anaerobic (QFR) respiratory chains. SQR from E. coli is homologous to mitochondrial complex II and has proven to be an excellent model system for structure/function studies of the enzyme. Both SQR and QFR from E. coli are tetrameric membrane-bound enzymes that couple succinate/fumarate interconversion with quinone/quinol reduction/oxidation. Both enzymes are capable of binding either ubiquinone or menaquinone, however, they have adopted different quinone binding sites where catalytic reactions with quinones occur. A comparison of the structures of the quinone binding sites in SQR and QFR reveals how the enzymes have adapted in order to accommodate both benzo- and napthoquinones. A combination of structural, computational, and kinetic studies of members of the complex II family of enzymes has revealed that the catalytic quinone adopts different positions in the quinone-binding pocket. These data suggest that movement of the quinone within the quinone-binding pocket is essential for catalysis.
Project description:The membrane-embedded quinol:fumarate reductase (QFR) in anaerobic bacteria catalyzes the reduction of fumarate to succinate by quinol in the anaerobic respiratory chain. The electron/proton-transfer pathways in QFRs remain controversial. Here we report the crystal structure of QFR from the anaerobic sulphate-reducing bacterium Desulfovibrio gigas (D. gigas) at 3.6?Å resolution. The structure of the D. gigas QFR is a homo-dimer, each protomer comprising two hydrophilic subunits, A and B, and one transmembrane subunit C, together with six redox cofactors including two b-hemes. One menaquinone molecule is bound near heme bL in the hydrophobic subunit C. This location of the menaquinone-binding site differs from the menaquinol-binding cavity proposed previously for QFR from Wolinella succinogenes. The observed bound menaquinone might serve as an additional redox cofactor to mediate the proton-coupled electron transport across the membrane. Armed with these structural insights, we propose electron/proton-transfer pathways in the quinol reduction of fumarate to succinate in the D. gigas QFR.
Project description:Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66-->Gln variant enzyme at 3.1-A resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.
Project description:In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site. Amino acids of this loop interact with both the substrate and a proton shuttle, potentially coordinating substrate binding and the proton shuttle protonation state. To assess the loop's role in catalysis, two threonine residues were mutated to alanine: QFR Thr-A244 (act-T; Thr-A254 in SQR), which hydrogen-bonds to the substrate at the active site, and QFR Thr-A234 (hinge-T; Thr-A244 in SQR), which is located at the hinge and hydrogen-bonds the proton shuttle. Both mutations impair catalysis and decrease substrate binding. The crystal structure of the hinge-T mutation reveals a reorientation between the FAD-binding and capping domains that accompanies proton shuttle alteration. Taken together, hydrogen bonding from act-T to substrate may coordinate with interdomain motions to twist the double bond of fumarate and introduce the strain important for attaining the transition state.
Project description: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. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.
Project description:Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H(+)/2e(-) quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction. In the complex II homolog quinol:fumarate reductase, it has been demonstrated that menaquinol oxidation requires at least one proton shuttle, but many of the remaining mechanistic details of menaquinol oxidation are not fully understood, and little is known about ubiquinone reduction. In the current study, structural and computational studies suggest that the sequential removal of the two menaquinol protons may be accompanied by a rotation of the naphthoquinone ring to optimize the interaction with a second proton shuttling pathway. However, kinetic measurements of site-specific mutations of quinol:fumarate reductase variants show that ubiquinone reduction does not use the same pathway. Computational docking of ubiquinone followed by mutagenesis instead suggested redundant proton shuttles lining the ubiquinone-binding site or from direct transfer from solvent. These data show that the quinone-binding site provides an environment that allows multiple amino acid residues to participate in quinone oxidoreduction. This suggests that the quinone-binding site in complex II is inherently plastic and can robustly interact with different types of quinones.
Project description:The epsilon-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic -proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation-reduction ('redox') midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.
Project description:Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.
Project description:A covalently bound flavin cofactor is predominant in the succinate-ubiquinone oxidoreductase (SQR; Complex II), an essential component of aerobic electron transport, and in the menaquinol-fumarate oxidoreductase (QFR), the anaerobic counterpart, although it is only present in approximately 10 % of the known flavoenzymes. This work investigates the role of this 8α-N3-histidyl linkage between the flavin adenine dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT calculations, classical molecular-dynamics simulations and quantum-mechanics calculations for Complex II:FAD and Complex II:FADH2 , with and without the covalent bond, were performed. It was observed that the covalent bond is essential for the active-center arrangement of the FADH2 /FAD cofactor. Removal of this bond causes a displacement of the isoalloxazine group, which influences interactions with the protein, flavin solvation, and possible proton-transfer pathways. Specifically, for the noncovalently bound FADH2 cofactor, the N1 atom moves away from the His-A365 and His-A254 residues and the N5 atom moves away from the glutamine-62A residue. Both of the histidine and glutamine residues interact with a chain of water molecules that cross the enzyme, which is most likely involved in proton transfer. Breaking this chain of water molecules could thereby compromise proton transfer across the two active sites of Complex II.
Project description:The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b(D) ring C (b(D)-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b(D)-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b(D)-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b(D) ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.