Existence of Two Types of Electrostatic Interaction in Reactions Catalyzed by Ferredoxin-NADP$ Oxidoreductase

Effects of salt and pH on diaphorase and NADP^$ photoreductionactivities were studied with broken spinach chloroplasts andpurified ferredoxin-NADP^$ oxidoreductase. Two types of electrostatic interactions, which regulate thereaction rate, were observed. One is the long-range electrostaticinteraction which determines the local concentrations of reactantsin the surface-mediated processes due to the change in the surfacepotential. In addition to the salt-induced change in the reactionrate, the pH optimum shift by salt was also remarkable: theoptimum pH in the diaphorase activity of chloroplasts shiftedto the more acidic pH region with an increase of salt concentration,while that of the membrane-free enzyme was not affected by salts. A more specific, short-range electrostatic interaction in reactionsbetween NADP(H) and ferredoxin-NADP^$ oxidoreductase was observed.This interaction became clearer when fixed charges on the membranesurface were masked by an addition of salts. Complete dissociationof the 2'-phosphate group of NADP(H) was necessary for its associationwith the enzyme. The eletrostatic attraction between the negativelychargedpart of NADPH and the positively-charged part of the enzyme(probably lysyl and arginyl residues) may play a role in theshort-range interaction. ¹Present address: Department of Agronomy, University of Kentucky,Lexington, Kentucky 40546, U.S.A. ²Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received February 21, 1983; Accepted March 17, 1984)     

色氨酸残基在磷酸烯醇式丙酮酸羧化酶催化功能中的作用

在pH7.5条件下,用NBS对PEP羧化酶中色氨酸残基进行共价修饰表明,PEP羧化酶中48个色氨酸残基均能被NBS修饰。用邹承鲁图解法求得,其中4个残基为酶表现催化活性所必需的。 PEP羧化酶的变构效应剂G6P、Gly及Mal分别与酶预保温后,再经NBS修饰,前两种处理中,同样浓度的NBS所用修饰的色氨酸残基数和处理后的残存酶活与对照相比有很大的差异,而用Mal处理的,两者与对照相差无几。     

Cryogenic and Laser Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven Enzyme Protochlorophyllide Oxidoreductase

The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of protochlorophyllide (Pchlide), a key step in chlorophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex.The light-driven enzyme protochlorophyllide oxidoreductase (POR)³ (EC 1.3.1.33) catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide) (see Fig. 1A) (1). This reaction is a key step in the synthesis of chlorophyll and leads to profound changes in the morphological development of photosynthetic organisms through modification and reorganization of plastid membranes (2, 3). In addition to POR, nonflowering land plants, algae, and cyanobacteria possess a light-independent Pchlide reductase, which consists of three separate subunits and allows these organisms to produce chlorophyllide in the dark (4). Together with DNA photolyase (5), POR is one of only two enzymes studied so far that exhibit a direct, natural requirement for light and because mixing strategies are no longer required to initiate the reaction, it is possible to trigger catalysis on very fast time scales and at cryogenic temperatures. Consequently, POR has proven to be an excellent model system for studying the role of protein dynamics in driving enzyme catalysis (1).Open in a separate windowFIGURE 1.The light-driven reduction of Pchlide. A, the trans addition of hydrogen across the C-17–C-18 double bond of Pchlide to form chlorophyllide (Chlide) in the chlorophyll biosynthesis pathway is catalyzed by the light-driven enzyme POR. B, shown is a three-dimensional model of the POR-catalyzed reaction based on the structural homology model of POR (26) and the proposed mechanism of hydride and proton transfer (8). Upon activation by light, a hydride is transferred to the C-17 position of Pchlide from the pro-S face of NADPH (shown in yellow), and the proton at the C-18 position is derived from Tyr-193 (shown in cyan). The conserved Lys-197 residue (shown in magenta) is proposed to decrease the pK_a of the Tyr to facilitate the proton transfer reaction.In the POR catalytic cycle, a ternary enzyme-NADPH-Pchlide complex is formed. Following light activation of this complex, a hydride ion is transferred from the pro-S face of NADPH to the C-17 atom of Pchlide (6, 7). The valence of the C-18 atom is satisfied by proton transfer, which is suggested to originate from an active site tyrosine residue (8). The catalytic cycle of POR has been analyzed through the trapping of intermediates at cryogenic temperatures. Following the initial light-driven reaction (9), there are a series of subsequent (slower) dark reactions (10, 11). The light-driven step involves hydride transfer from NADPH to form a charge transfer complex, which then facilitates protonation of the pigment intermediate during the first of the “dark” reactions (12). Moreover, through laser activation of catalysis, we have shown that both of these H-transfer reactions proceed by quantum mechanical tunneling coupled to motions in the enzyme-substrate complex on the submicrosecond time scale (13). The final dark steps in the reaction cycle involve a series of ordered product release and cofactor binding steps linked to conformational changes in the enzyme (10, 11, 14). Ultrafast measurements have uncovered spectral changes on the picosecond timescale that are likely to represent conformational changes prior to Pchlide reduction (15–19). Previous excitation of POR with a laser pulse leads to a more efficient conformation of the active site and an enhancement in the catalytic efficiency of the enzyme (18).POR is a member of a large family of enzymes known as short chain dehydrogenases/reductases (SDR). These are single domain NAD(P)⁺- or NAD(P)H-binding oxidoreductases that exist generally as dimers or tetramers (20). A number of SDR enzymes (e.g. carbonyl reductase, alcohol dehydrogenase, and dihydrofolate reductase) have been good model systems for studying the dynamics linked to enzyme catalysis (21–23). This family of enzymes has been amenable to studies of biological H-tunneling (24–26), and in particular the unique light-activated properties of POR make it an excellent system for studying mechanisms of H-transfer and dynamics in this family of enzymes.Structures of several SDR family members are available, and these have enabled the construction of a homology model of POR from Synechocystis (27). This model comprises a central parallel β-sheet of seven β-strands, surrounded by nine α-helices, with an additional unique 33-residue insertion between the fifth and sixth β-sheets. The NADPH cofactor binds within the N-terminal region of the enzyme, which contains a common nucleotide-binding motif with a tight βαβ fold, termed the Rossmann fold (27). Importantly, a Tyr and a Lys residue are both absolutely conserved throughout all members of the SDR family and are critical for catalysis in a number of enzymes (28–31). A common mechanism has been proposed for this group of enzymes, involving a Tyr-X-X-X-Lys motif. The Lys residue in this motif is presumed to facilitate proton donation from the Tyr hydroxyl group to substrate through favorable perturbation of the hydroxyl group pK_a (8, 31). In POR, multiple turnover assays have also indicated that these Tyr and Lys residues are important for activity (8, 32, 33), leading to a proposed mechanism that involves proton transfer from the conserved Tyr residue to the C-18 position of Pchlide (8) (Fig. 1B). The close proximity of the Lys residue is thought to allow the deprotonation step to occur at physiological pH by lowering the apparent pK_a of the phenolic group of the Tyr (8). However, confirmation of the exact role of these conserved residues has been compromised by the limited levels of activity observed in previous studies of the variant enzymes (8, 32, 33), and a detailed evaluation of the role of the active site Tyr and Lys residues on the chemical steps (i.e. hydride and proton transfer) has not been reported. We address this deficiency in the current work by analyzing a number of site-specific mutant forms altered at Tyr-193 and Lys-197 in a thermophilic POR from Thermosynechococcus elongatus BP-1. This was achieved using steady-state (multiple turnover) and laser photoexcitation (single turnover) methods and by trapping transient reaction intermediates by fluorescence spectroscopy performed at cryogenic temperatures.     

Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site

Abstract: The pH dependency of the binding of ligands to adenosine A_2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5'- N -ethylcarboxamidoadenosine, adenosine, 3'-deoxyadenosine, 5'-deoxyadenosine, inosine, and N ⁶-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2'-deoxyadenosine, although 2'-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2'-hydroxyl group of adenosine agonists.     

Crystal Structure of the Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase Catalytic Complex (ChlN/ChlB)2

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL₂ bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)₂ complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)₂ complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel β-sheet surrounded by α-helices. The (ChlN/ChlB)₂ crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)₂(ChlL₂)₂ complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.     

Light-Induced Damage of Histidine Residues in Photosystem II Reaction Center D1/D2/cyt b559 Complex

Histidine residue content of photosystem Ⅱ reaction center D1/D2/cytochrome b559 complex decreased by about 26% after illumination. The result suggests that some histidine residues are damaged by illumination. The damage of histidine residues may be related to the changes of the spectra properties during the incubation in the dark following preillumination of the reaction center complex.     

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys⁸²) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys⁸⁷ was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys⁸². These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.     

Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

The pathway of autotrophic CO₂ fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO₂-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either ¹⁴CO₂ or [¹⁴C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO₂ fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.     

Disulfide Bonds within the C2 Domain of RAGE Play Key Roles in Its Dimerization and Biogenesis

Background

The receptor for advanced glycation end products (RAGE) on the cell surface transmits inflammatory signals. A member of the immunoglobulin superfamily, RAGE possesses the V, C1, and C2 ectodomains that collectively constitute the receptor''s extracellular structure. However, the molecular mechanism of RAGE biogenesis remains unclear, impeding efforts to control RAGE signaling through cellular regulation.

Methodology and Result

We used co-immunoprecipitation and crossing-linking to study RAGE oligomerization and found that RAGE forms dimer-based oligomers. Via non-reducing SDS-polyacrylamide gel electrophoresis and mutagenesis, we found that cysteines 259 and 301 within the C2 domain form intermolecular disulfide bonds. Using a modified tripartite split GFP complementation strategy and confocal microscopy, we also found that RAGE dimerization occurs in the endoplasmic reticulum (ER), and that RAGE mutant molecules without the double disulfide bridges are unstable, and are subjected to the ER-associated degradation.

Conclusion

Disulfide bond-mediated RAGE dimerization in the ER is the critical step of RAGE biogenesis. Without formation of intermolecular disulfide bonds in the C2 region, RAGE fails to reach cell surface.

Significance

This is the first report of RAGE intermolecular disulfide bond.     

Role of an Essential Acyl Coenzyme A Carboxylase in the Primary and Secondary Metabolism of Streptomyces coelicolor A3(2)

Two genes, accB and accE, that form part of the same operon, were cloned from Streptomyces coelicolor A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of accB and accE in Escherichia coli and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an accB null mutant and the thiostrepton growth dependency of a tipAp accB conditional mutant confirmed that AccB is essential for S. coelicolor viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109–3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites.     

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

Summary: Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO₂ to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO₂ to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.     

Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A

Our earlier finding that the activity of protein phosphatase 2A from rat brain is inhibited by micromolar concentrations of the dithiol cross-linking reagent phenylarsine oxide (PAO) has encouraged the hypothesis that the catalytic subunit (PP2Ac) of PP2A contains one or more pairs of closely-spaced (vicinal) thiol pairs that may contribute to regulation of the enzyme. The results of the present study demonstrate using immobilized PAO-affinity chromatography that PP2Ac from rat brain formed stable DTT-sensitive adducts with PAO with or without associated regulatory subunits. In addition, a subset of the PAO-binding vicinal thiols of PP2Ac was readily oxidized to disulfide bonds in vitro. Importantly, a small fraction of PP2Ac was still found to contain disulfide bonds after applying stringent conditions designed to prevent protein disulfide bond formation during homogenization and fractionation of the brains. These findings establish the presence of potentially regulatory and redox-active PAO-binding vicinal thiols on the catalytic subunit of PP2A and suggest that a population of PP2Ac may contain disulfide bonds in vivo.     

Role of Histidine Residues in Agonist and Antagonist Binding Sites of A1 Adenosine Receptor

Abstract: The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N⁶-[adenine-2,8-³H, ethyl-2-³H]phenylisopropyladenosine ([³H]R-PIA) and antagonist 8-cyclopentyl-1,3-[³H]-dipropylxanthine ([³H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [³H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [³H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A₁ adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (K_D) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand. The pattern of protective action of R-PIA, DPCPX, adenosine, and guanylylimidodiphosphate in DEP treatment and the displacement curves of radiolabeled agonist and antagonist by both unlabeled ligands indicated that the interaction site for agonist and antagonist binding is the same, although the complete mechanisms for recognition and binding differ.     

Involvement of NADH:Acceptor Oxidoreductase and Butyryl Coenzyme A Dehydrogenase in Reversed Electron Transport during Syntrophic Butyrate Oxidation by Syntrophomonas wolfei

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting Syntrophomonas wolfei and the methanogen Methanospirillum hungatei, and a reversed electron transport system in S. wolfei was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H₂ generation. The metabolic activity of butyrate-oxidizing S. wolfei cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of S. wolfei, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of S. wolfei. The results suggest that syntrophic oxidation of butyrate in S. wolfei involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered “bifurcating” hydrogenase in Thermotoga maritima and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.Butyrate is fermented to methane and CO₂ by syntrophic communities in which a methanogenic partner organism maintains a low hydrogen partial pressure to allow the oxidation of butyrate to acetate (19, 20, 29). Only under such conditions can butyrate-oxidizing bacteria such as Syntrophomonas wolfei gain energy from the latter reaction in a range of approximately −20 kJ per mol of butyrate, which is just sufficient to support microbial growth (29). It was postulated that S. wolfei has to invest some of the ATP that is formed in the acetate kinase reaction during the β-oxidation of butyrate into an ATP-driven reversed electron transport in order to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH (34).Experimental evidence for the involvement of a proton gradient and of ATPase activity in this process was obtained with intact cell suspensions (36), and it was hypothesized that menaquinone-7 could play an essential role in this reaction (36). This would imply that membrane-bound enzymes similar to complex I of the aerobic respiratory chain, i.e., NADH dehydrogenase (NDH), operate in reverse to reduce NAD⁺ with butyrate electrons.Another option for a reversed electron transport during butyrate oxidation and hydrogen formation in S. wolfei could be a reversal of the so-called Buckel-Thauer reaction. In this reaction that was described for ethanol-acetate fermentation by Clostridium kluyveri, electrons from NADH are disproportionated to reduce both crotonyl-CoA and ferredoxin simultaneously. The reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase/electron-transfer flavoprotein (Bcd/EtfAB) complex (13, 18). Very recently, another “bifurcating” electron pathway has been described for an NADH- and ferredoxin-coaccepting di-iron hydrogenase complex in Thermotoga maritima (30). Here, electrons from NADH and from ferredoxin are combined to produce hydrogen, and the genome sequence of S. wolfei has been shown to contain candidate genes for such a three-component hydrogenase complex (30). Nonetheless, the energetic situation of syntrophic butyrate oxidation is basically different from that of ethanol or glucose degradation: electrons arise at comparably positive redox potentials, i.e., at −125 mV/−10 mV (12, 28) and −250 mV, and there is no oxidation step involved that could be coupled directly with ferredoxin reduction.In the present study, we report that butyrate oxidation by S. wolfei cell suspensions can be inhibited by trifluoperazine (TPZ), an antitubercular agent that has been shown to inhibit type II NADH:menaquinone oxidoreductase NDH-2 in Mycobacterium tuberculosis (40), and that a TPZ-sensitive NADH:acceptor oxidoreductase activity can be measured in cell extracts of S. wolfei cells. This enzyme system and a butyryl-CoA dehydrogenase were enriched by anion-exchange chromatography, and the obtained proteins were identified by peptide mass fingerprinting.     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

Induction of the Inactivation and Degradation of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Maize Leaves by Freezing and Thawing

When frozen leaves of 24-day-old maize (Zea mays L.) plant werethawed on moist filter paper at 26°C (freeze-thaw treatment)several enzymes, including phosphoenolpyruvate carboxylase (PEPC)and ribulose-1,5-bisphosphate carboxylase (RuBPC), were rapidlyinactivated and degraded. The kinetics of the inactivation anddegradation were pseudo first-order, and the halftimes for inactivationof PEPC and RuBPC were 3.2 and 2.4 min, respectively. The effectof the freeze-thaw treatment on the inactivation and degradationdiffered among various enzymes: the residual activities of RuBPC,PEPC, hydroxypyruvate reductase, Cyt c oxidase, NADP-malic enzymeand a-mannosidase 10 min after the start of the thawing treatmentwere 7, 16, 54, 64, 97 and 98% of the initial respective levels.Thirty min after the starting of thawing treatment, the amountsof total soluble protein, the large subunit of RuBPC, the smallsubunit of RuBPC, the PEPC subunit and the NADP-malic enzymesubunit had fallen to 61, 2, 16, 8, and 66% of the initial respectiveamounts. The effect of freeze-thaw treatment on PEPC was greater in oldleaves than in young leaves. There was a steady increase ofthe rate of degradation of PEPC by freeze-thaw treatment asplants aged from 6 to 24 days. These results are discussed inthe context of protein degradation in plant cells. (Received August 9, 1993; Accepted January 10, 1994)