Ubiquinol Binding Research Articles

Kinetic and structural characterisation of the ubiquinol-binding site and oxygen reduction by the trypanosomal alternative oxidase

The alternative oxidase (AOX) is a monotopic di‑iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs.In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in Vmax and catalytic efficiency whilst some also affected the Km for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC50 values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle. SignificanceThe alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production.

Insights into the ubiquinol/dioxygen binding and proton relay pathways of the alternative oxidase

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein which catalyzes the four-electron reduction of dioxygen to water by ubiquinol. Although we have recently determined the crystal structure of Trypanosoma brucei AOX (TAO) in the presence and absence of ascofuranone (AF) derivatives (which are potent mixed type inhibitors) the mechanism by which ubiquinol and dioxygen binds to TAO remain inconclusive. In this article, ferulenol was identified as the first competitive inhibitor of AOX which has been used to probe the binding of ubiquinol. Surface plasmon resonance reveals that AF is a quasi-irreversible inhibitor of TAO whilst ferulenol binding is completely reversible. The structure of the TAO-ferulenol complex, determined at 2.7 Å, provided insights into ubiquinol binding and has also identified a potential dioxygen molecule bound in a side-on conformation to the diiron center for the first time.

Searching for the low affinity ubiquinone binding site in cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase is one of three respiratory oxygen reductases in the aerobic respiratory chain of Escherichia coli. The generally accepted model of catalysis assumes that cyt bo3 contains two distinct ubiquinol binding sites: (i) a low affinity (QL) site which is the traditional substrate binding site; and (ii) a high affinity (QH) site where a “permanently” bound quinone acts as a cofactor, taking two electrons from the substrate quinol and passing them one-by-one to the heme b component of the enzyme which, in turn, transfers them to the heme o3/CuB active site. Whereas the residues at the QH site are well defined, the location of the QL site remains unknown. The published X-ray structure does not contain quinone, and substantial amounts of the protein are missing as well. A recent bioinformatics study by Bossis et al. [Biochem J. (2014) 461, 305–314] identified a sequence motif G163EFX3GWX2Y173 as the likely QL site in the family of related quinol oxidases. In the current work, this was tested by site-directed mutagenesis. The results show that these residues are not important for catalytic function and do not define the QL substrate binding site.

Novel Inhibitors for a Novel Binding Site in Respiratory Complex III.

A new binding site and potential novel inhibitors of the respiratory complex III are described. The site is located at the opposite side of the enzyme with respect to ubiquinol binding site (Qo site), and distinctly different from both Qo and Qi sites (hence designated as Non-Q binding site, NQ). NQ site binding pocket extends up close to Phe90 residue, an internal switch (LH switch) that regulates electron transfer between heme bL and heme bH of the low potential redox chain. Docking studies and molecular dynamics simulations of different molecules to the NQ site revealed potential ligands which exhibit a novel inhibitory effect for bc1 complex by switching the LH switch to "off" conformation, thereby shutting down electron transfer in the low potential redox chain. Moreover, the novel inhibitors have lower binding affinity for both Qo and Qi sites, and hence do not interfere with binding of the natural ligands to those sites. The inhibitory activity of those novel ligands in bc1 complex is suggested to promote the production of reactive oxygen species (ROS) at the Qo site. Hence those ligands are potential candidates for designing new "mitocan" drugs.

Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex.

Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton-coupled electron transfer from quinol to iron–sulfur cluster through a bridging hydrogen bond from histidine that stabilizes the reaction complex.

Peroxidase activity of cytochrome bd from Escherichia coli

Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H2O2 concentration is linear up to the concentration of 8 mM. At higher concentrations of H2O2, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling. Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b(558), and H2O2 is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological role for humans and animals.

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia ( Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc 1 complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A 3 in a fashion similar to the Q o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q i and the Q o sites of the fungal cytochrome bc 1 complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron–sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc 1 complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q o and Q i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc 1 complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q i and Q o sites. Moreover, crystal structure of chicken cytochrome bc 1 complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q i and Q o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc 1 inhibitor that acts at both of the active sites of this enzyme.

Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border, 2002–2005

The combination of atovaquone and proguanil (Malarone™, GlaxoSmithKline) has been established as an effective drug of choice not only for treatment but also for prophylaxis of multi-drug resistant Plasmodium falciparum malaria in travellers. However, several cases of resistance against Malarone™ have already been reported in some parts of Africa, and many of the cases are associated with mutations at codon 268 of cytochrome b gene in mitochondrial genome of P. falciparum. On the contrary, in Asian countries, only limited numbers of clinical failures with the drug has so far been reported, and no mutations could be found at the codon 268. To maintain the usefulness of Malarone™ in travel medicine, monitoring of the effectiveness of the drug is needed. In this study, mutations of atovaquone binding site on the cytochrome b were investigated by PCR amplification and sequencing of the gene (1131 bp length) in the parasites from patients at the Hospital for Tropical Diseases Mahidol University in Bangkok, Thailand, in 2002 through 2005. Twenty-five blood samples were from complicated falciparum malaria patients and another 25 were from non-complicated falciparum patients who were infected in multi-drug resistant areas in Thai-Myanmar border. Forty-seven out of 50 samples were successfully analyzed, and no mutation was observed in putative ubiquinol (Q0) binding site 1 and 2. In fact, they showed identically conserved nucleotide sequences, as was reported by our previous study conducted in the same area in 1999. We may conclude from these findings that Malarone™ is still effective and recommended for treatment and prophylaxis for travellers to Mekong region.

Growth inhibition of Toxoplasma gondii and Plasmodium falciparum by nanomolar concentrations of 1-hydroxy-2-dodecyl-4(1H)quinolone, a high-affinity inhibitor of alternative (type II) NADH dehydrogenases.

Both apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum lack type I NADH dehydrogenases (complex I) but instead carry alternative (type II) NADH dehydrogenases, which are absent in mammalian cells and are thus considered promising antimicrobial drug targets. The quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) was recently described as a high-affinity inhibitor of fungal alternative NADH dehydrogenases in enzymatic assays, probably by interfering with the ubiquinol binding site of the enzyme. We describe here that HDQ effectively inhibits the replication rates of P. falciparum and T. gondii in tissue culture. The 50% inhibitory concentration (IC50) of HDQ for T. gondii was determined to be 2.4+/-0.3 nM with a growth assay based on vacuole sizes and 3.7+/-1.4 nM with a growth assay based on beta-galactosidase activity. Quantification of the P. falciparum replication rate using a fluorometric assay revealed an IC50 of 14.0+/-1.9 nM. An important feature of the HDQ structure is the length of the alkyl side chain at position 2. Derivatives with alkyl side chains of C6, C8, C12 (HDQ), and C14 all displayed excellent anti-T. gondii activity, while a C5 derivative completely failed to inhibit parasite replication. A combined treatment of T. gondii-infected cells with HDQ and the antimalarial agent atovaquone, which blocks the ubiquinol oxidation site of cytochrome b in complex III, resulted in synergism, with a calculated fractional inhibitory concentration of 0.16 nM. Interference of the mitochondrial ubiquinone/ubiquinol cycle at two different locations thus appears to be a highly effective strategy for inhibiting parasite replication. HDQ and its derivatives, particularly in combination with atovaquone, represent promising compounds with a high potential for antimalarial and antitoxoplasmal therapy.

Probing the Role of E272 in Quinol Oxidation of Mitochondrial Complex III

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of complex III catalysis, but the molecular basis of this process is still not clear. E272 of the conserved cytochrome b PEWY motif has been suggested as a ligand and proton acceptor for ubiquinol oxidation at center P. We introduced the two replacement mutations, E272D and E272Q, into the mitochondrially encoded cytochrome b gene by biolistic transformation to study their effects on substrate binding and catalysis. Both substitutions resulted in a lower ubiquinol cytochrome c reductase activity and affect the KM for ubiquinol. The E272 carboxylate stabilizes stigmatellin binding, and in accordance, both variants are resistant to stigmatellin. Large structural changes in the cofactor environment as well as in the binding pocket can be excluded. The mutations do not perturb the midpoint potentials of the heme groups. The sensitivity toward the respective distal and proximal niche inhibitors HDBT and myxothiazol is retained. However, both mutations provoke subtle structural alterations detected by redox FTIR. They affect binding and oxidation of ubiquinol, and they promote electron short-circuit reactions resulting in production of reactive oxygen species. The aspartate substitution modifies the environment of the reduced Rieske protein as monitored by EPR. Both variants alter the pH dependence of the enzyme activity. Diminished activity at low pH coincides with the loss of one protonatable group with a pKa of approximately 6.2 compared to three pKa values in the wild type, supporting the role of E272 in proton transfer. The conserved glutamate appears to influence the accurate formation of the enzyme-substrate complex and to govern the efficiency of catalysis.

The Iron-Sulfur Cluster of the Rieske Iron-Sulfur Protein Functions as a Proton-exiting Gate in the Cytochrome bc1 Complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.

Kinetics of Electron Transfer within Cytochrome bc1and Between Cytochrome bc1and Cytochrome c

In this minireview an overview is presented of the kinetics of electron transfer within the cytochrome bc (1) complex, as well as from cytochrome bc (1) to cytochrome c. The cytochrome bc (1) complex (ubiquinone:cytochrome c oxidoreductase) is an integral membrane protein found in the mitochondrial respiratory chain as well as the electron transfer chains of many respiratory and photosynthetic bacteria. Experiments on both mitochondrial and bacterial cyatochrome bc (1) have provided detailed kinetic information supporting a Q-cycle mechanism for electron transfer within the complex. On the basis of X-ray crystallographic studies of cytochrome bc (1), it has been proposed that the Rieske iron-sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c (1). A new method was developed to study electron transfer within cytochrome bc (1) using a binuclear ruthenium complex to rapidly photooxidize cytochrome c (1). The rate constant for electron transfer from the iron-sulfur center to cytochrome c (1) was found to be 80,000 s(-1), and is controlled by the dynamics of conformational changes in the iron-sulfur protein. Moreover, a linkage between the conformation of the ubiquinol binding site and the conformational dynamics of the iron-sulfur protein has been discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method has also been developed to measure electron transfer from cytochrome c (1) to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc (1) and cytochrome c.

Ischemia–reperfusion injury in the aged heart: role of mitochondria

The aged heart sustains greater injury during ischemia and reperfusion compared to the adult heart. Aging decreases oxidative phosphorylation and the activity of complexes III and IV only in interfibrillar mitochondria (IFM) that reside among the myofibrils, whereas subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. The peptide subunit composition of complexes III and IV is intact in aging. The aging defect in complex IV is in the inner membrane lipid environment. The defect in complex III is within the ubiquinol binding site of the cytochrome b subunit. Following ischemia, in the aged heart both SSM and IFM sustain additional decreases in complex III and complex IV activity. In contrast to the aging defect, with ischemia the subunits of complex IV appear to be damaged. Ischemia inactivates the iron–sulfur peptide subunit in complex III. Mitochondria are the major source of the reactive oxygen species that are generated during myocardial ischemia. Complex III is the major site of mitochondrial oxyradical production during ischemia in the adult heart. The role of complex III in the oxidative damage sustained by the aged heart during ischemia, as well as the potential contribution of aging defects in electron transport to ischemic damage in the aged heart, deserves further study. We propose that following ischemic damage to the electron transport chain, the production and release of reactive oxygen species increases from mitochondria in the aged heart, leading to additional damage during reperfusion.

Aging defect at the Q O site of complex III augments oxyradical production in rat heart interfibrillar mitochondria

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q O) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q O site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.

Nonoxidizable ubiquinol derivatives that are suitable for the study of the ubiquinol oxidation site in the cytochrome bc1 complex

Recent X-ray crystallographic analyses of the mitochondrial cytochrome bc 1 complex show ubiquinone binding at the Q i site, but attempts to show binding of ubiquinol or ubiquinone at the Q o site have been unsuccessful, even though the binding of noncompetitive Q o site inhibitors near the putative ubiquinol binding pocket is well established. We speculate that ubiquinol binds transiently to the Q o site only when both heme b L and the iron sulfur cluster are in the oxidized form, an experimental condition difficult to obtain since ubiquinol will be oxidized once bound to the site. Stable binding at the Q o site might be achieved by a nonoxidizable ubiquinol-like compound. For this purpose, the isomers 2,3,4-trimethoxy-5-decyl-6-methyl-phenol (TMDMP) and 2,3,4-trimethoxy-5-methyl-6-decyl-phenol (TMMDP) were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1, 4-benzoquinol (Q 0C 10) by controlled methylation and separated by TLC and HPLC. The structures of TMDMP and TMMDP were established by 1H- 13C-two-dimensional NMR. Both are competitive inhibitors of the cytochrome bc 1 complex, with TMDMP being the stronger one. Preliminary results suggest that TMDMP binds tightly enough to make X-ray crystallography of inhibitor– bc 1 complex co-crystals feasible. The binding site of TMDMP does not overlap with the binding sites of stigmatellin, MOA-stilbene (MOAS), undecylhydroxydioxobenzothiazole (UHDBT) and myxothaizol.

Interactions of quinone with the iron–sulfur protein of the bc1 complex: is the mechanism spring-loaded?

Since available structures of native bc 1 complexes show a vacant Q o-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme–substrate complex at the Q o-site. This is formed by binding of both ubiquinol (QH 2) and the dissociated oxidized iron–sulfur protein (ISP ox). A binding constant of ∼14 can be estimated from the displacement of E m or p K for quinone or ISP ox, respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme–product complex is also formed at the Q o-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E m of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E m reflects a binding constant of ∼4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q o-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.

Role of protonatable groups of bovine heart bc(1) complex in ubiquinol binding and oxidation.

The pH dependence of the initial reaction rate catalyzed by the isolated bovine heart ubiquinol-cytochrome c reductase (bc1 complex) varying decylbenzoquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group with pKa = 5.7 +/- 0.2, while the inhibition constant (Ki) for DB decreased 22 and 2.8 times when groups with pKa = 5.2 +/- 0.6 and 7.7 +/- 0.2, respectively, were protonated. This suggests stabilization of the protonated form of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pKa approximately 5.7 group blocked catalysis, indicating its role in proton transfer. The kinetic model assumed that the deprotonation of two groups (pKa values of 7.5 +/- 0.03 and approximately 9.2) decreases the catalytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) cluster. The protonation of the pKa approximately 7.5 group also decreased the reaction rate by 80-86%, suggesting its role as acceptor of a proton from ubiquinol. The lack of effect on the Km for DBH when the pKa 7.5-7.7 group is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Qo site. The possible relationship of the pKa 5.2-5.7 and pKa 7.5-7.7 groups with Glu272 of cytochrome b and His161 of the Fe-S protein is discussed.

New sequence data enable modelling of the fungal alternative oxidase and explain an absence of regulation by pyruvate

Respiratory rates involving the alternative oxidase (AO) were studied in mitochondria from Tapesia acuformis. There was no evidence for regulation by pyruvate, in contrast with plant AO. The site of interaction of pyruvate with the plant AO is a conserved cysteine. The primary sequence was obtained for AO from Magnaporthe grisea and compared with four published sequences for fungal AO. In all cases this cysteine was absent. Sequence data were obtained for the C-terminal domain of a further five fungal AOs. In this region the fungal sequences were all consistent with a four-helix, di-iron binding structure as in the ferritin-fold family. A molecular model of this domain was deduced from the structure of Δ-9 desaturase. This is in general agreement with that developed for plant AOs, despite very low sequence identity between the two kingdoms. Further modelling indicated an appropriate active site for binding of ubiquinol, required in the AO redox reaction.

Tryptophan-136 in subunit II of cytochrome bo3 from Escherichia coli may participate in the binding of ubiquinol.

In the cytochrome c oxidases, the role of subunit II is to provide the electron entry site into the enzyme. This subunit contains both the binding site for the substrate, cytochrome c, and the CuA redox center, which is initially reduced by cytochrome c. Cytochrome bo3 and other quinol oxidases that are members of the heme-copper oxidase superfamily have a homologous subunit II, but the CuA site is absent, as is the docking site for cytochrome c. Speculation that subunit II in the quinol oxidases may also be important as an electron entry site is supported by the demonstration several years ago that a photoreactive substrate analogue, azido-Q, covalently labeled subunit II in cytochrome bo3. In the current work, a sequence alignment of subunit II of heme-copper quinol oxidases is used as a guide to select conserved residues that might be important for the binding of ubiquinol to cytochrome bo3. Results are presented for point mutants in 24 different residue positions in subunit II. The membrane-bound enzymes were examined by optical spectroscopy and by determining the activity of ubiquinol-1 oxidase. In each case, the Km for ubiquinol-1 was determined as a measure of possible perturbation to a quinol binding site. The only mutant that had a noticeably altered Km for ubiquinol-1 was W136A, in which the Km was about sixfold increased. Thus, W136 may be at or close to a substrate (ubiquinol)-binding site in cytochrome bo3. In the cytochrome c oxidases, the equivalent tryptophan (W121 in Paracoccus denitrificans) has been identified as the "electron entry site".

Gene linkage and steady state RNAs suggest trans-splicing may be associated with a polycistronic transcript in Schistosoma mansoni

Spliced leader (SL) trans-splicing generates the 5′ end of mature mRNAs through the addition of a small exon to pre-mRNAs in some flagellates (kinetoplastida and euglenoids) and metazoans (nematodes and flatworms). Although SL addition in the kinetoplastida and a subset of nematode genes serves to resolve multicistronic mRNAs into monocistronic, capped mRNAs, information regarding the functional significance of trans-splicing in flatworms is limited. We describe here the identification and characterization of a closely linked gene upstream from the trans-spliced enolase gene in the flatworm Schistosoma mansoni. This gene produces a non- trans-spliced mRNA encoding a ubiquinol binding protein, UbCRBP, that is a component of the ubiquinol-cytochrome C reductase complex. The distance between the UbCRBP polyadenylation site and the enolase trans-splice acceptor site is exceptionally short, only 54 nucleotides. Primer extension (5′ RACE), RT-PCR, and RNase mapping have identified steady state, cis-spliced RNAs which significantly overlap both the UbCRBP and enolase genes. These transcripts contain the 5′ ends of mature UbCRBP mRNAs, extend through UbCRBP, across the intergenic region, and a significant distance 3′ into the enolase gene. Interestingly, the close linkage between the UbCRBP and enolase genes is conserved in a second flatworm, Fasciola hepatica, which also trans-splices the downstream enolase gene. Taken together, the role of SL addition in resolving multicistronic transcripts in both C. elegans and the kinetoplastida, the conservation of UbCRBP/enolase gene linkage in two divergent trematodes, and the multicistronic organization of schistosome UbCRBP/enolase RNAs are consistent with the suggestion that these two genes are likely to be cotranscribed and that trans-splicing in flatworms may be associated with polycistronic transcripts.