Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples Its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.

Flavocytochrome b2-based enzymatic method of L-lactate assay in food products.

L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b 2 (EC 1.1.2.3; FC b 2) isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB) reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ = 525 nm and the linear range is observed in the interval 0.005-0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 µM of L-lactate), simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b 2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b 2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

Flavin-containing heme enzymes.

There are many examples of oxidative enzymes containing both flavin and heme prosthetic groups that carry out the oxidation of their substrate. For the purpose of this article we have chosen five systems. Two of these, the L-lactate dehydrogenase flavocytochrome b(2) and cellobiose dehydrogenase, carry out the catalytic chemistry at the flavin group. In contrast, the remaining three require activation of dioxygen at the heme group in order to accomplish substrate oxidation, these being flavohemoglobin, a nitric oxide dioxygenase, and the mono-oxygenases nitric oxide synthase and flavocytochrome P450 BM3, which functions as a fatty acid hydroxylase. In the light of recent advances we will describe the structures of these enzymes, some of which share significant homology. We will also discuss their diverse and sometimes controversial catalytic mechanisms, and consider electron transfer processes between the redox cofactors in order to provide an overview of this fascinating set of enzymes.

Protein sensing with engineered protein nanopores.

The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the α-hemolysin (αHL) protein pore has been used extensively for the detection, characterization, and analysis of polypeptides because this protein nanopore is highly robust, versatile, and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered αHL pores. Here, we provide a detailed protocol for the preparation of αHL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins.

Dynamics of flavin semiquinone protolysis in L-alpha-hydroxyacid-oxidizing flavoenzymes--a study using nanosecond laser flash photolysis.

The reactions of the flavin semiquinone generated by laser-induced stepwise two-photon excitation of reduced flavin have been studied previously (El Hanine-Lmoumene C & Lindqvist L. (1997) Photochem Photobiol 66, 591-595) using time-resolved spectroscopy. In the present work, we have used the same experimental procedure to study the flavin semiquinone in rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase domain of flavocytochrome b(2) FDH, two homologous flavoproteins belonging to the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes. For both proteins, pulsed laser irradiation at 355 nm of the reduced enzyme generated initially the neutral semiquinone, which has rarely been observed previously for these enzymes, and hydrated electron. The radical evolved with time to the anionic semiquinone that is known to be stabilized by these enzymes at physiological pH. The deprotonation kinetics were biphasic, with durations of 1-5 micros and tens of microseconds, respectively. The fast phase rate increased with pH and Tris buffer concentration. However, this increase was about 10-fold less pronounced than that reported for the neutral semiquinone free in aqueous solution. pK(a) values close to that of the free flavin semiquinone were obtained from the transient protolytic equilibrium at the end of the fast phase. The second slow deprotonation phase may reflect a conformational relaxation in the flavoprotein, from the fully reduced to the semiquinone state. The anionic semiquinone is known to be an intermediate in the flavocytochrome b(2) catalytic cycle. In light of published kinetic studies, our results indicate that deprotonation of the flavin radical is not rate-limiting for the intramolecular electron transfer processes in this protein.

Structural evidence for the functional importance of the heme domain mobility in flavocytochrome b2.

Yeast flavocytochrome b(2) (Fcb2) is an L-lactate:cytochrome c oxidoreductase in the mitochondrial intermembrane space participating in cellular respiration. Each enzyme subunit consists of a cytochrome b(5)-like heme domain and a flavodehydrogenase (FDH) domain. In the Fcb2 crystal structure, the heme domain is mobile relative to the tetrameric FDH core in one out of two subunits. The monoclonal antibody B2B4, elicited against the holoenzyme, recognizes only the native heme domain in the holoenzyme. When bound, it suppresses the intramolecular electron transfer from flavin to heme b(2), hence cytochrome c reduction. We report here the crystal structure of the heme domain in complex with the Fab at 2.7 A resolution. The Fab epitope on the heme domain includes the two exposed propionate groups of the heme, which are hidden in the interface between the domains in the complete subunit. The structure discloses an unexpected plasticity of Fcb2 in the neighborhood of the heme cavity, in which the heme has rotated. The epitope overlaps with the docking area of the FDH domain onto the heme domain, indicating that the antibody displaces the heme domain in a movement of large amplitude. We suggest that the binding sites on the heme domain of cytochrome c and of the FDH domain also overlap and therefore that cytochrome c binding also requires the heme domain to move away from the FDH domain, so as to allow electron transfer between the two hemes. Based on this hypothesis, we propose a possible model of the Fcb2.cytochrome c complex. Interestingly, this model shares similarity with that of the cytochrome b(5) x cytochrome c complex, in which cytochrome c binds to the surface around the exposed heme edge of cytochrome b(5). The present results therefore support the idea that the heme domain mobility is an inherent component of the Fcb2 functioning.

L-lactate dehydrogenation in flavocytochrome b2: a first principles molecular dynamics study.

First principles molecular dynamics studies on active-site models of flavocytochrome b2 (L-lactate : cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidation reaction, a still-debated issue. In the calculated enzyme-substrate model complex, the L-lactate alpha-OH hydrogen is hydrogen bonded to the active-site base H373 Nepsilon, whereas the Halpha is directed towards flavin N5, suggesting that the reaction is initiated by alpha-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidation led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to flavin mononucleotide, without intermediates, but with alpha-OH proton abstraction preceding Halpha transfer and a calculated free energy barrier (12.1 kcal mol(-1)) consistent with that determined experimentally (13.5 kcal mol(-1)). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a number of flavoenzymes. Namely, they highlighted the role of: (a) the flavin mononucleotide-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin reduction; (b) an active site water molecule belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction.

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base.

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.

Potentiometric and further kinetic characterization of the flavin-binding domain of Saccharomyces cerevisiae flavocytochrome b2. Inhibition by anions binding in the active site.

Saccharomyces cerevisiae flavocytochrome b2 (L-lactate:cytochrome c oxido reductase, EC 1.1.2.3) is a homotetramer, with FMN and protoheme IX binding on separate domains. The flavin-binding domains form the enzyme tetrameric core, while the cytochrome b2 domains appear to be mobile around a hinge region (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 867-863). The enzyme catalyzes electron transfer from L-lactate to cytochrome c, or to nonphysiological acceptors such as ferricyanide, via FMN and heme b2. The kinetics of this multistep reaction are complex. In order to clarify some of its aspects, the tetrameric FMN-binding domain (FDH domain) has been independently expressed in Escherichia coli (Balme, A., Brunt, C. E., Pallister, R., Chapman, S. K., and Reid, G. A. (1995) Biochem. J. 309, 601-605). We present here an additional characterization of this domain. In our hands, it has essentially the same ferricyanide reductase activity as the holo-enzyme. The comparison of the steady-state kinetics with ferricyanide as acceptor and of the pre-steady-state kinetics of flavin reduction, as well as the kinetic isotope effects of the reactions using L-2-[2H]lactate, indicates that flavin reduction is the limiting step in lactate oxidation. During the oxidation of the reduced FDH domain by ferricyanide, the oxidation of the semiquinone is much faster than the oxidation of two-electron-reduced flavin. This order of reactivity is reversed during flavin to heme b2 transfer in the holo-enzyme. Potentiometric studies of the protein yielded a standard redox potential for FMN at pH 7.0, E(o)7, of -81 mV, a value practically identical to the published value of -90 mV for FMN in holo-flavocytochrome b2. However, as expected from the kinetics of the oxidative half-reaction, the FDH domain was characterized by a significantly destabilized flavin semiquinone state compared with holo-enzyme, with a semiquinone formation constant K of 1.25-0.64 vs 33.5, respectively (Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., and Bertrand, P. (1998) Biochemistry, 37, 12761-12771). As in the holo-enzyme, the semiquinone state in the FDH domain is significantly stabilized by the reaction product, pyruvate. We also studied the inhibition exerted in the steady and pre steady states by the reaction product pyruvate and by anions (bromide, chloride, phosphate, acetate), with respect to both flavin reduction and reoxidation. The results indicate that these compounds bind to the oxidized and the two-electron-reduced forms of the FDH domain, and that excess L-lactate also binds to the two-electron-reduced form. These findings point to the existence of a common or strongly overlapping binding site. A comparison of the effect of the anions on WT and R289K holo-flavocytochromes b2 indicates that invariant R289 belongs to this site. According to literature data, it must also be present in other members of the family of L-2-hydroxy acid-oxidizing enzymes.

Electron flow in multicenter enzymes: theory, applications, and consequences on the natural design of redox chains.

In protein film voltammetry, a redox enzyme is directly connected to an electrode; in the presence of substrate and when the driving force provided by the electrode is appropriate, a current flow reveals the steady-state turnover. We show that, in the case of a multicenter enzyme, this signal reports on the energetics and kinetics of electron transfer (ET) along the redox chain that wires the active site to the electrode, and this provides a new strategy for studying intramolecular ET. We propose a model which takes into account all the enzyme's redox microstates, and we prove it useful to interpret data for various enzymes. Several general ideas emerge from this analysis. Considering the reversibility of ET is a requirement: the usual picture, where ET is depicted as a series of irreversible steps, is oversimplified and lacks the important features that we emphasize. We give justification to the concept of apparent reduction potential on the time scale of turnover and we explain how the value of this potential relates to the thermodynamic and kinetic properties of the system. When intramolecular ET does not limit turnover, the redox chain merely mediates the driving force provided by the electrode or the soluble redox partner, whereas when intramolecular ET is slow, the enzyme behaves as if its active active site had apparent redox properties which depend on the reduction potentials of the relays. This suggests an alternative to the idea that redox chains are optimized in terms of speed: evolutionary pressure may have resulted in slowing down intramolecular ET in order to tune the enzyme's "operating potential".

Altered substrate specificity in flavocytochrome b2: structural insights into the mechanism of L-lactate dehydrogenation.

Flavocytochrome b(2) from Saccharomyces cerevisiae is a l-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301, 829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate l-mandelate than it is with l-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 A resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 A resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavages are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism.

Yeast flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate; because of the wealth of structural and mechanistic information available, this enzyme has served as the model for the family of flavoproteins catalyzing oxidation of alpha-hydroxy acids. Primary deuterium and solvent isotope effects have now been used to analyze the effects of mutating the active site residue Tyr254 to phenylalanine. Both the V(max) and the V/K(lactate) values decrease about 40-fold in the mutant enzyme. The primary deuterium isotope effects on the V(max) and the V/K(lactate) values increase to 5.0, equivalent to the intrinsic isotope effect for the wild-type enzyme. In addition, both the V(max) and the V/K(lactate) values exhibit solvent isotope effects of 1.5. Measurement of the solvent isotope effect with deuterated lactate establishes that the primary and solvent isotope effects arise from the same chemical step, consistent with concerted cleavage of the lactate OH and CH bonds. The pH dependence of the mutant enzyme is not significantly different from that of the wild-type enzyme; this is most consistent with a requirement that the side chain of Tyr254 be uncharged for catalysis. The results support a hydride transfer mechanism for the mutant protein and, by extension, wild-type flavocytochrome b(2) and the other flavoproteins catalyzing oxidation of alpha-hydroxy acids.

Carbanion versus hydride transfer mechanisms in flavoprotein-catalyzed dehydrogenations.

The present understanding of the mechanisms by which flavoproteins oxidize amino acid or hydroxy acids to the respective imino or keto acids is reviewed. The observation that many of these enzymes catalyze the elimination of HBr or HCl from the appropriate beta-halogenated substrate was long considered evidence for a carbanion intermediate. Recent structural and mechanistic studies are not compatible with the intermediacy of carbanions in the reactions catalyzed by d-amino acid oxidase and flavocytochrome b(2). In contrast, the data are most consistent with mechanisms involving direct hydride transfer.

Tom40 protein import channel binds to non-native proteins and prevents their aggregation.

Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.