Insights into the catalytic mechanism of type VI sulfide:quinone oxidoreductases

Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redox-active centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class.     

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 Å resolution for native unbound protein in space group P4₂2₁2 . The decylubiquinone-bound structure and the Cys160Ala variant structure were subsequently determined to 2.3 Å and 2.05 Å resolutions, respectively, in space group P6₂22  . The enzymatic reaction catalyzed by sulfide:quinone oxidoreductase includes the oxidation of sulfide compounds H₂S, HS⁻, and S²⁻ to soluble polysulfide chains or to elemental sulfur in the form of octasulfur rings; these oxidations are coupled to the reduction of ubiquinone or menaquinone. The enzyme comprises two tandem Rossmann fold domains and a flexible C-terminal domain encompassing two amphipathic helices that are thought to provide for membrane anchoring. The second amphipathic helix unwinds and changes its orientation in the hexagonal crystal form. The protein forms a dimer that could be inserted into the membrane to a depth of approximately 20 Å. It has an endogenous flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the N-terminal domain. Several wide channels connect the FAD cofactor to the exterior of the protein molecule; some of the channels would provide access to the membrane. The ubiquinone molecule is bound in one of these channels; its benzoquinone ring is stacked between the aromatic rings of two conserved Phe residues, and it closely approaches the isoalloxazine moiety of the FAD cofactor. Two active-site cysteine residues situated on the re side of the FAD cofactor form a branched polysulfide bridge. Cys356 disulfide acts as a nucleophile that attacks the C4A atom of the FAD cofactor in electron transfer reaction. The third essential cysteine Cys128 is not modified in these structures; its role is likely confined to the release of the polysulfur product.     

Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.     

Sulfide oxidation in gram-negative bacteria by expression of the sulfide-quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone.

The oxidation of sulfide was studied in recombinant bacteria expressing the sulfide-quinone reductase gene (sqr) from Rhodobacter capsulatus. Sulfide was oxidized by the Escherichia coli strain W3110 harboring the sqr construct (pKKSQ) under anaerobic conditions and nitrate was utilized as a terminal electron acceptor. Following the oxidation, elemental sulfur and nitrite were produced as the final reaction products. This activity was retained in the membrane preparation and was sensitive towards antimycin A, stigmatellin, and azide. As a consequence of the ubiquinone deficiency, this activity was markedly decreased. In additon, by recovery of ubiquinone, the oxidation was also restored to rates similar to those of the wild-type strain. These results indicate that sulfide oxidation in this strain occurs via the quinone pool in vivo, and that this sulfide-quinone reductase (SQR) in particular utilizes ubiquinone as a more appropriate electron acceptor than menaquinone or demetylmenaquinone. To our knowledge, this is the first study to show a direct interaction between SQR and ubiquinone in cells. When expressed in Pseudomonas putida and Rhizobium meliloti, the SQR conferred on these organisms the ability to oxidize sulfide as well as E. coli in vivo.     

Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.     

硫醌氧化还原酶研究进展

硫化物是一种广泛分布的有毒物质。硫醌氧化还原酶是生物体硫化物代谢的一种关键酶。对该酶的发现与分布、序列特征、催化机制、催化特性、三维结构和生理功能等几个方面进行概述,并对该酶未来研究方向进行展望。     

The oligomeric state of the Caldivirga maquilingensis type III sulfide:Quinone Oxidoreductase is required for membrane binding

Sulfide:quinone oxidoreductase (SQR) is a monotopic membrane flavoprotein present in all domains of life, with multiple roles including sulfide detoxification, homeostasis and energy generation by providing electrons to respiratory or photosynthetic electron transport chains. A type III SQR from the hyperthermophilic archeon Caldivirga maquilingensis has been previously characterized, and its C-terminal amphipathic helices were demonstrated to be responsible for membrane binding. Here, the oligomeric state of this protein was experimentally evaluated by size exclusion chromatography, native gels and crosslinking, and found to be a monomer-dimer-trimer equilibrium. Remarkably, mutant and truncated variants unable to bind to the membrane are able to maintain their oligomeric association. Thus, unlike other related monotopic membrane proteins, the region involved in membrane binding does not influence oligomerization. Furthermore, by studying heterodimers between the WT and mutants, it was concluded that membrane binding requires an oligomer with at least two copies of the protein with intact C-terminal amphipathic helices. A structural homology model of the C. maquilingensis SQR was used to define the flavin- and quinone-binding sites. CmGly12, CmGly16, CmAla77 and CmPro44 were determined to be important for flavin binding. Unexpectedly, CmGly299 is only important for quinone reduction despite its proximity to bound FAD. CmPhe337 and CmPhe362 are also important for quinone binding apparently by direct interaction with the quinone ring, whereas CmLys359, postulated to hydrogen bond to the quinone, seems to have a more structural role. The results presented differentiate the Type III CmSQR from some of its counterparts classified as Type I, II and V.     

Preparation and structure of the charge-transfer intermediate of the transmembrane redox catalyst DsbB

Disulfide bond formation is a critical step in the folding of many secretory proteins. In bacteria, disulfide bonds are introduced by the periplasmic dithiol oxidase DsbA, which transfers its catalytic disulfide bond to folding polypeptides. Reduced DsbA is reoxidized by ubiquinone Q8, catalyzed by inner membrane quinone reductase DsbB. Here, we report the preparation of a kinetically stable ternary complex between wild-type DsbB, containing all essential cysteines, Q8 and DsbA covalently bound to DsbB. The crystal structure of this trapped DsbB reaction intermediate exhibits a charge-transfer interaction between Q8 and the Cys44 in the DsbB reaction center providing experimental evidence for the mechanism of de novo disulfide bond generation in DsbB.     

Mutagenesis of the redox-active disulfide in mercuric ion reductase: catalysis by mutant enzymes restricted to flavin redox chemistry

Mercuric reductase, a flavoenzyme that possess a redox-active cystine, Cys135Cys140, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, we have constructed mutants lacking a redox-active disulfide by eliminating Cys135 (Ala135Cys140), Cys140 (Cys135Ala140), or both (Ala135Ala140). Additionally, we have made double mutants that lack Cys135 (Ala135Cys139Cys140) or Cys140 (Cys135Cys139Ala140) but introduce a new Cys in place of Gly139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. These differences are manifested in a 23-nm range in enzyme-bound FAD lambda max values, an 80-nm range in thiolate to flavin charge-transfer absorbance maxima, and a ca. 100-mV range in FAD reduction potential. Preliminary evidence for the Ala135Cys139Cys140 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala135Cys140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. For these activities, there is a linear correlation between log kappa cat and enzyme-bound FAD reduction potential. In a sensitive Hg(II)-mediated enzyme-bound FADH2 reoxidation assay, all mutant enzymes were able to undergo at least one catalytic event at rates 50-1000-fold slower than that of the wild-type enzyme. We have also observed the reduction of Hg(II) by free FADH2. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. We conclude that the Cys135 and Cys140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.     

嗜酸氧化亚铁硫杆菌的谷胱甘肽还原酶的结构模建和底物分子对接

极端环境微生物嗜酸氧化亚铁硫杆菌的谷胱甘肽还原酶(GR)可能在它的抵抗极端酸性,有毒和氧化性的生物浸出环境中发挥至关重要的作用.通过同源模建技术和分子动力学模拟,它的一个三维结构被构建,优化和检验了.获得的结构被进一步用于搜索绑定位点,跟辅因子黄素腺嘌呤二核苷酸(FAD)和底物谷胱甘肽(GSSG)进行分子柔性对接,并以此识别关健残基.对接结果显示,位于活性残基Cys42和Cys47之间的二硫键夹在FAD的活性位点和底物GSSG的二硫键之间.它们之间的距离非常靠近,这跟底物反应机理的初始步骤的情况十分一致.相互作用能表明8个酶中残基Cys42,Cys47,GIu443B,Glu444B,His438B,Ser14,Thr447B和Lys51是固定或激活GSSG的关键残基,这跟以前的实验事实相吻合.此外,根据相互作用能我们还新发现7个重要残基(Arg449B,Pro439B,Thr440B,Thr310,Va143,Gly46 and Va148).所有这些残基在其它物种中的相应物中也都是保守的.这些结果有助于进一步的实验研究和理解其催化机理,进而揭示这种细菌的抗毒机理,服务于工业应用.     

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.     

Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase

The first step in the mitochondrial sulfide oxidation pathway is catalyzed by sulfide quinone oxidoreductase (SQR), which belongs to the family of flavoprotein disulfide oxidoreductases. During the catalytic cycle, the flavin cofactor is intermittently reduced by sulfide and oxidized by ubiquinone, linking H₂S oxidation to the electron transfer chain and to energy metabolism. Human SQR can use multiple thiophilic acceptors, including sulfide, sulfite, and glutathione, to form as products, hydrodisulfide, thiosulfate, and glutathione persulfide, respectively. In this study, we have used transient kinetics to examine the mechanism of the flavin reductive half-reaction and have determined the redox potential of the bound flavin to be −123 ± 7 mV. We observe formation of an unusually intense charge-transfer (CT) complex when the enzyme is exposed to sulfide and unexpectedly, when it is exposed to sulfite. In the canonical reaction, sulfide serves as the sulfur donor and sulfite serves as the acceptor, forming thiosulfate. We show that thiosulfate is also formed when sulfide is added to the sulfite-induced CT intermediate, representing a new mechanism for thiosulfate formation. The CT complex is formed at a kinetically competent rate by reaction with sulfide but not with sulfite. Our study indicates that sulfide addition to the active site disulfide is preferred under normal turnover conditions. However, under pathological conditions when sulfite concentrations are high, sulfite could compete with sulfide for addition to the active site disulfide, leading to attenuation of SQR activity and to an alternate route for thiosulfate formation.     

Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases

The anaerobically expressed fumarate reductase and aerobically expressed succinate dehydrogenase from Escherichia coli comprise two different classes of succinate:quinone oxidoreductases (SQR), often termed respiratory complex II. The X-ray structures of both membrane-bound complexes have revealed that while the catalytic/soluble domains are structurally similar the quinone binding domains of the enzyme complexes are significantly different. These results suggest that the anaerobic and aerobic forms of complex II have evolved different mechanisms for electron and proton transfer in their respective membrane domains.     

Use of a site-directed triple mutant to trap intermediates: demonstration that the flavin C(4a)-thiol adduct and reduced flavin are kinetically competent intermediates in mercuric ion reductase

A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates. The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+. The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes.     

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.     

Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus: CONFORMATIONAL CHANGES DURING CATALYSIS AND IMPLICATIONS FOR THE CATALYTIC AND INHIBITORY MECHANISMS*

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys¹⁰² functions as the catalytic residue and Cys⁶⁸ as the resolving Cys that forms a disulfide bond with Cys¹⁰². Cys⁷⁸, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys¹⁰². We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.     

Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activity

The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mM. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (K(m) = 6.4 microm) after the addition of sulfide (K(m) = 23 microm) only in the presence of cyanide (K(m) = 2.6 mM). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme.     

Proton uptake in the reaction center mutant L210DN from Rhodobacter sphaeroides via protonated water molecules

The reaction center (RC) of Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, QB (the secondary quinone electron acceptor), to form quinol, QBH2. Asp210 in the L-subunit has been shown to be a catalytic residue in this process. Mutation of Asp210 to Asn leads to a deceleration of reoxidation of QA- in the QA-QB --> QAQB- transition. Here we determined the structure of the Asp210 to Asn mutant to 2.5 A and show that there are no major structural differences as compared to the wild-type protein. We found QB in the distal position and a chain of water molecules between Asn210 and QB. Using time-resolved Fourier transform infrared (trFTIR) spectroscopy, we characterized the molecular reaction mechanism of this mutant. We found that QB- formation precedes QA- oxidation even more pronounced than in the wild-type reaction center. Continuum absorbance changes indicate deprotonation of a protonated water cluster, most likely of the water chain between Asn210 and QB. A detailed analysis of wild-type structures revealed a highly conserved water chain between Asp210 or Glu210 and QB in Rb. sphaeroides and Rhodopseudomonas viridis, respectively.     

Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli.

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.