Plasticity of the Quinone-binding Site of the Complex II Homolog Quinol:Fumarate Reductase

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.     

The Quinol:Fumarate Oxidoreductase from the Sulphate Reducing Bacterium Desulfovibrio gigas: Spectroscopic and Redox Studies

The membrane bound fumarate reductase (FRD) from the sulphate-reducer Desulfovibrio gigas was purified from cells grown on a fumarate/sulphate medium and extensively characterized. The FRD is isolated with three subunits of apparent molecular masses of 71, 31, and 22 kDa. The enzyme is capable of both fumarate reduction and succinate oxidation, exhibiting a higher specificity toward fumarate (K _m for fumarate is 0.02 and for succinate 2 mM) and a reduction rate 30 times faster than that for oxidation. Studies by Visible and EPR spectroscopies allowed the identification of two B-type haems and the three iron–sulphur clusters usually found in FRDs and succinate dehydrogenases: [2Fe-2S]^2+/1+ (S1), [4Fe-4S]^2+/1+ (S2), and [3Fe-4S]^1+/0 (S3). The apparent macroscopic reduction potentials for the metal centers, at pH 7.6, were determined by redox titrations: –45 and –175 mV for the two haems, and +20 and –140 mV for the S3 and S1 clusters, respectively. The reduction potentials of the haem groups are pH dependent, supporting the proposal that fumarate reduction is associated with formation of the membrane proton gradient. Furthermore, co-reconstitution in liposomes of D. gigas FRD, duroquinone, and D. gigas cytochrome bd shows that this system is capable of coupling succinate oxidation with oxygen reduction to water.     

A Soluble Fumarate Reductase in Trypanosoma brucei Procyclic Trypomastigotes

ABSTRACT. The enzyme NADH-fumarate reductase associated with the membrane fraction of Trypanosoma brucei procyclic trypomastigotes, can be solubilized by more than 50% when increasing the ionic strength to the equivalent of 150 mM KCI. The apparent K_Ms for NADH (125 μM) and fumarate (50 μM) remain close to those previously reported for the membrane-bound form of this enzyme. Other electron acceptors (i.e. oxygen or cytochrome c) appear to accept electrons in the absence of fumarate (K_M for cytochrome c = 50 μM). The drug L-092,201 (Merck, Sharp and Dohme Research Laboratories, Rahway, NJ), an inhibitor of the membrane-bound fumarate reductase, also blocked the solubilized enzyme. Given the relatively high ionic strength of the intracellular environment we propose that, in vivo, the enzyme fumarate reductase is in the mitochondrial matrix or in the soluble fraction of another intracellular compartment.     

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.     

The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat⁺ strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat⁺ strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat⁺ strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat⁺ strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat⁺ strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat⁺ strain colonized chickens at wild-type levels.Campylobacter jejuni causes approximately two million cases of bacterial gastroenteritis in the United States annually (34). Humans are most often infected due to cross-contamination resulting from improper handling of poultry (27), which is the natural habitat of C. jejuni (28). The eradication of C. jejuni from poultry flocks is an important goal in reducing the number of campylobacteriosis cases.C. jejuni can rely solely on catabolism of small organic acids and amino acids as a carbon and energy source, and the products of this catabolism are used for glycolysis and the tricarboxylic acid (TCA) cycle (15, 29). Fumarate and succinate are key intermediates in the TCA cycle, and the interconversion of these compounds is a vital process in organisms that use the TCA cycle for central carbon metabolism. C. jejuni encodes a complete oxidative TCA cycle, which converts TCA intermediates (carboxylic acids) to CO₂, ATP, and reducing equivalents. One of the conversion steps, oxidation of succinate to fumarate, forms a reducing equivalent and is required for a complete cycle. Reduction of fumarate to succinate also occurs as part of the reductive TCA cycle, and this carbon fixation pathway has been proposed to be utilized by ɛ-proteobacteria found in deep-sea hydrothermal vents (3). C. jejuni encodes many of the reversible enzymes necessary for the reductive TCA cycle, including 2-oxoglutarate ferredoxin oxidoreductase (encoded by oorDABC) and pyruvate carboxylase (encoded by pycA and pycB) (29); however, C. jejuni does not encode an ATP citrate lyase, which is required for full cyclic reductive carboxylation (3). The fumarate-succinate interconversion is also involved in respiration (11), and fumarate has specifically been implicated as an electron acceptor that is an alternative to oxygen in other ɛ-proteobacteria (5, 17).C. jejuni encodes an enzyme which is annotated as a fumarate reductase (Cj0408 to Cj0410) and an enzyme which is annotated as a succinate dehydrogenase (Cj0437 to Cj0439) (29). Both of these enzymes are part of a large family of proteins called the succinate:quinone oxidoreductases (SQRs). These compounds are membrane-bound enzymes that either catalyze the two-electron oxidation of succinate to the two-electron reduction of quinone/quinol or, in the reverse direction, couple the oxidation of quinol/quinone to the reduction of fumarate to succinate. The amino acid sequence, however, does not dictate the in vivo function (18), and in characterized organisms like Escherichia coli both enzymes are able to reduce fumarate and oxidize succinate, albeit with a preference for one substrate (6, 21).The SQRs can be divided into three distinct classes based on function, all of which have similar subunit compositions and primary amino acid sequences. Class 1 SQRs couple the oxidation of succinate to the reduction of a high-redox-potential quinone like ubiquinone in vivo. Class 2 SQRs are the quinol:fumarate reductases, which couple the oxidation of menaquinol to the reduction of fumarate. And class 3 SQRs couple the oxidation of succinate to the reduction of a low-potential quinone, such as menaquinone, in vivo (11). Although each class has shared motifs, the in vivo function of an SQR enzyme cannot be resolved based on the primary sequence and must be determined experimentally. Fumarate reductase (Frd) activity has been reported to occur in the particulate fraction of C. jejuni cell lysates, and addition of formate to whole cells increased Frd activity (38), which implies that there is an active electron transport pathway. However, C. jejuni is unable to utilize fumarate as an alternative electron acceptor under anaerobic conditions (37, 41). C. jejuni can also use succinate as an electron donor to a respiratory quinone (12), which has been identified as either a menaquinone-6 or methylmenaquinone-6 (4). Yet succinate oxidation via menaquinone is an endergonic reaction; succinate has a redox midpoint potential (E_m) of 30 mV, and menaquinone is more electronegative (E_m = −80 mV). Although succinate oxidation coupled to menaquinone reduction would be an “uphill” reaction, class 3 SQRs can catalyze this reaction. Studies of gram-positive bacteria belonging to the genus Bacillus, as well as studies of sulfate-reducing bacteria, have shown that oxidation of succinate through menaquinone is driven by reverse transmembrane electron transport (18, 36, 45), and it is hypothesized that C. jejuni behaves similarly. The C. jejuni Frd enzyme contains three subunits, FrdC, FrdA, and FrdB, and the gene order in the operon is similar to that in Wolinella succinogenes (16, 19) and Helicobacter pylori (1, 9, 40). Based on Frd enzymes of other bacteria, FrdC (Cj0408) is the membrane anchor and diheme cytochrome b, FrdA (Cj0409) is a flavoprotein where the reduction of fumarate to succinate occurs, and FrdB (Cj0410) is an Fe-S protein (29). The succinate dehydrogenase of C. jejuni is also composed of three subunits, SdhABC encoded by Cj0437 to Cj0439 (29). SdhA is annotated as a succinate dehydrogenase flavoprotein subunit, SdhB is a putative succinate dehydrogenase Fe-S protein, and SdhC is a putative succinate dehydrogenase subunit C. According to ClustalW pairwise alignment, FrdA and SdhA of C. jejuni share 29% identity, FrdB and SdhB share 18% identity, and FrdC and SdhC share 13% identity.A better understanding of the C. jejuni TCA cycle may help identify metabolic pathways that are crucial to C. jejuni''s ability to thrive in poultry. The roles of the C. jejuni fumarate reductase and succinate dehydrogenase in the TCA cycle and respiration were investigated. Both enzymes contribute to the total fumarate reductase activity. We determined that the protein annotated as the fumarate reductase functions as the sole succinate dehydrogenase and that this enzyme is required for full colonization of chickens by C. jejuni. The sdh operon has been misannotated as the enzyme that it encodes exhibits no succinate dehydrogenase activity, as has recently been reported to be the case for the annotated succinate dehydrogenase of W. succinogenes (14).     

Cloning and Sequencing of the Gene Encoding the Soluble Fumarate Reductase from Saccharomyces cerevisiae

A gene of the soluble fumarate reductase (FRDS) that binds FADnon-covalently was cloned by polymerase chain reaction (PCR)using degenerate oligonucleotides designed from partial aminoacid sequences of highly purified enzyme. The nucleotide sequenceof a 0.99-kb amplified product was found to be nearly identicalto a partial sequence of an open reading frame (ORF) previouslyreported (EMBL database accession number S-30830). Accordingto the sequence in the EMBL database, we cloned 1.7-kb fragmentcontaining entire sequence of this ORF by PCR and found thatthis fragment contained a perfect match to the 0.99-kb sequenceamplified with the degenerate primers. From these results, weconcluded that this ORF is the FRDS gene. The amino acid sequencesof the regions involved in the non-covalent binding of FAD andthe active site, which are conserved among the flavoproteinsubunits of membrane-bound fumarate reductase and succinatedehydrogenase, were found in FRDS. However, unlike the membrane-boundenzymes, FRDS did not contain the histidine residue that covalentlybinds the isoalloxazine ring of FAD at or near the correspondingposition. FRDS showed high homology to the product of S. cerevisiaeOSM1 gene which was reported to be required for growth in hypertonicmedia.     

Estimating the Stochastic Bifurcation Structure of Cellular Networks

High throughput measurement of gene expression at single-cell resolution, combined with systematic perturbation of environmental or cellular variables, provides information that can be used to generate novel insight into the properties of gene regulatory networks by linking cellular responses to external parameters. In dynamical systems theory, this information is the subject of bifurcation analysis, which establishes how system-level behaviour changes as a function of parameter values within a given deterministic mathematical model. Since cellular networks are inherently noisy, we generalize the traditional bifurcation diagram of deterministic systems theory to stochastic dynamical systems. We demonstrate how statistical methods for density estimation, in particular, mixture density and conditional mixture density estimators, can be employed to establish empirical bifurcation diagrams describing the bistable genetic switch network controlling galactose utilization in yeast Saccharomyces cerevisiae. These approaches allow us to make novel qualitative and quantitative observations about the switching behavior of the galactose network, and provide a framework that might be useful to extract information needed for the development of quantitative network models.     

Proteins of the Inner Membrane of Escherichia coli: Changes in Composition Associated with Anaerobic Growth and Fumarate Reductase Amber Mutation

The inner membrane fractions of Escherichia coli grown anaerobically and aerobically were isolated, and their proteins were compared by electrophoresis in polyacrylamide gels. To maximimize the differences between the preparations, the anaerobic cultures were grown on complex medium with added glucose, but glucose was omitted from the aerobic cultures to prevent catabolite repression. The pattern of bands in the two types of preparation differed considerably, and changes in approximately 20 components were observed. In particular, the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. Mutants lacking fumarate reductase were isolated, and inner membrane preparations of an frd amber mutant were deficient in a major component of 75,000 daltons and possibly a minor one of 87,500 daltons. The former was also present in greater amounts in anaerobic preparations and could represent a fumarate reductase subunit.     

Anaerobic Expression of Escherichia coli Succinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the P_FRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.     

延迟遗传调控网络稳定性及分支

本文主要研究了延迟遗传调控网络的局部稳定性和该网络的Hopf分支存在条件．延迟遗传调控网络是无穷维系统,此类系统在平衡点线性化后的特征方程为超越方程。通过对此超越方程进行研究,得到了系统系数不同时的系统稳定的条件及相关结论,又进一步说明了此系统的Hopf分支存在条件．最后,举一个例子进行了数值仿真验证了所得到的结论．     

Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.     

Quinol oxidation by c-type cytochromes: structural characterization of the menaquinol binding site of NrfHA

Membrane-bound cytochrome c quinol dehydrogenases play a crucial role in bacterial respiration by oxidizing menaquinol and transferring electrons to various periplasmic oxidoreductases. In this work, the menaquinol oxidation site of NrfH was characterized by the determination of the X-ray structure of Desulfovibrio vulgaris NrfHA nitrite reductase complex bound to 2-heptyl-4-hydroxyquinoline-N-oxide, which is shown to act as a competitive inhibitor of NrfH quinol oxidation activity. The structure, at 2.8-Å resolution, reveals that the inhibitor binds close to NrfH heme 1, where it establishes polar contacts with two essential residues: Asp89, the residue occupying the heme distal ligand position, and Lys82, a strictly conserved residue. The menaquinol binding cavity is largely polar and has a wide opening to the protein surface. Coarse-grained molecular dynamics simulations suggest that the quinol binding site of NrfH and several other respiratory enzymes lie in the head group region of the membrane, which probably facilitates proton transfer to the periplasm. Although NrfH is not a multi-span membrane protein, its quinol binding site has several characteristics similar to those of quinone binding sites previously described. The data presented here provide the first characterization of the quinol binding site of the cytochrome c quinol dehydrogenase family.     

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

Biogenic Aldehyde Metabolism in Rat Brain: Subcellular Distribution of Aldose Reductase and Valproate-Sensitive Aldehyde Reductase

Reductase activity towards two aldose substrates has been examined in subcellular fractions prepared from rat brain. The reduction of glucuronate, which is sensitive to inhibition by the anticonvulsant drug sodium valproate, corresponds to the major high-Km aldehyde reductase in brain. Xylose reduction that is insensitive to valproate inhibition has characteristics consistent with the activity of aldose reductase (EC 1.1.1.21). Both enzymes are predominantly localized in the cytosolic fraction. The significance of the location of these two reductases is discussed in relation to the compartmentation of catecholamine metabolism in brain.     

Crystal Structure of Red Chlorophyll Catabolite Reductase: Enlargement of the Ferredoxin-Dependent Bilin Reductase Family

The key steps in the degradation pathway of chlorophylls are the ring-opening reaction catalyzed by pheophorbide a oxygenase and sequential reduction by red chlorophyll catabolite reductase (RCCR). During these steps, chlorophyll catabolites lose their color and phototoxicity. RCCR catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite. RCCR appears to be evolutionarily related to the ferredoxin-dependent bilin reductase (FDBR) family, which synthesizes a variety of phytobilin pigments, on the basis of sequence similarity, ferredoxin dependency, and the common tetrapyrrole skeleton of their substrates. The evidence, however, is not robust; the identity between RCCR and FDBR HY2 from Arabidopsis thaliana is only 15%, and the oligomeric states of these enzymes are different. Here, we report the crystal structure of A. thaliana RCCR at 2.4 Å resolution. RCCR forms a homodimer, in which each subunit folds in an α/β/α sandwich. The tertiary structure of RCCR is similar to those of FDBRs, strongly supporting that these enzymes evolved from a common ancestor. The two subunits are related by noncrystallographic 2-fold symmetry in which the α-helices near the edge of the β-sheet unique in RCCR participate in intersubunit interaction. The putative RCC-binding site, which was derived by superimposing RCCR onto biliverdin-bound forms of FDBRs, forms an open pocket surrounded by conserved residues among RCCRs. Glu154 and Asp291 of A. thaliana RCCR, which stand opposite each other in the pocket, likely are involved in substrate binding and/or catalysis.     

Human Brain Aldehyde Reductases: Relationship to Succinic Semialdehyde Reductase and Aldose Reductase

Human brain contains multiple forms of aldehyde-reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH-dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase ("SSA reductase") specifically reduces succinic semialdehyde (SSA) to produce gamma-hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions.     

Fumarate: Multiple functions of a simple metabolite

Although much is now known about fumarate metabolism, our knowledge of some aspects of its biological function remain far from comprehensive. In this short review we begin with an introductory overview of the role of fumarate in both plant and non-plant systems. We next highlight the relative importance of fumarate in relation to cell type and circumstance in contrast to other chemically similar organic acids. Considerable cumulative evidence is suggestive of a role for fumarate in pH regulation during nitrate assimilation and that fumarate has similar effects as malate during stomatal movement. Indeed it is currently difficult to separate the biological function of fumarate from malate under certain circumstances. However, in other cases this can be easily performed. This physiological complexity notwithstanding it remains possible that the engineering of fumarate metabolism may provide opportunities to improve plant growth and performance.     

Quinol:fumarate oxidoreductases and succinate:quinone oxidoreductases: phylogenetic relationships, metal centres and membrane attachment.

A comprehensive phylogenetic analysis of the core subunits of succinate:quinone oxidoreductases and quinol:fumarate oxidoreductases is performed, showing that the classification of the enzymes as type A to E based on the type of the membrane anchor fully correlates with the specific characteristics of the two core subunits. A special emphasis is given to the type E enzymes, which have an atypical association to the membrane, possibly involving anchor subunits with amphipathic helices. Furthermore, the redox properties of the SQR/QFR proteins are also reviewed, stressing out the recent observation of redox-Bohr effect upon haem reduction, observed for the Desulfovibrio gigas and Rhodothermus marinus enzymes, which indicates a direct protonation event at the haems or at a nearby residue. Finally, the possible contribution of these enzymes to the formation/dissipation of a transmembrane proton gradient is discussed, considering recent experimental and structural data.