[27] Flavocytochrome b2 or l-lactate cytochrome c reductase from yeast

Labeyrie, Françoise; Baudras, Alain; Lederer, Florence

doi:10.1016/s0076-6879(78)53030-9

Cited by 106 publications

(83 citation statements)

References 28 publications

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“…Flavocytochrome b2 was prepared either from commercial bakers' yeast (United Yeast Co. Ltd, Menstrie, Scotland) or from GR20 transformants as described previously [21]. Phenylmethylsulphonyl fluoride (1 mM) was present throughout the preparation to yield the intact enzyme [21].…”

Section: Enzyme Preparationmentioning

confidence: 99%

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Probing the active site of flavocytochrome b₂ by site‐directed mutagenesis

Reid

White

Black

et al. 1988

European Journal of Biochemistry

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The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiue) has recently been solved at 0.24-nm resolution in Flavins and Jlavoproteins, Walter de Gruyter, Berlin, pp. 123-1311. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene.The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of Llactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced k,,, from 190 f 3 s -l (25"C, pH 7.5) to 4.3 & 0.1 s-'. This substitution had, however, no discernable effect on K, for lactate (0.54 f 0.03 mM for the mutant compared with 0.49 0.03 mM for the wild-type enzyme). Arg376 was expected to be essential for productive binding and orientation of L-lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s-' to 160 s-') and K,,, (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of L-lactate oxidation.Flavocytochrome b2 (L-lactate: cytochrome c oxidoreductase) from bakers' yeast is a tetramer of identical subunits with M , = 57500 [l]. Each subunit consists of two functionally distinct domains, one containing flavin mononucleotide (FMN), the other a protoheme IX group [2]. The protein is a soluble component of the mitochondria1 intermembrane space [3] where it catalyzes the oxidation of Llactate to pyruvate and transfers electrons directly to cytochrome c [2]. The crystal structure of flavocytochrome h2 has been solved [4, 51 allowing identification of the substrate binding site adjacent to the flavin. The structural information can be correlated with mechanistic models resulting from studies carried out in solution. It is thus possible to propose the following roles for various active-site residues (see also [a : a) Arg376 and Tyr143 bind and orient the substrate through electrostatic and hydrogen-bond interactions with the carboxylate group. b) His373 acts as a general base, abstracting the C, hydrogen as a proton resulting in carbanion formation [7]. c) Tyr254 also acts as a general base, deprotonating the substrate hydroxyl and thus facilitating electron transfer from the carbanion to the flavin. d) Lys349 facilitates electron transfer by stabilizing the N1 anion of the reduced flavin. e) Ala306 is located some distance from the active site for lactate oxidation within a disordered loop region. The mobility of this region appears to be important fo...

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Section: Enzyme Preparationmentioning

confidence: 99%

“…Phenylmethylsulphonyl fluoride (1 mM) was present throughout the preparation to yield the intact enzyme [21]. Enzyme preparations were stored under nitrogen at 4°C as precipitates in 70% (NH4)2S04.…”

Section: Enzyme Preparationmentioning

confidence: 99%

Probing the active site of flavocytochrome b₂ by site‐directed mutagenesis

Reid

White

Black

et al. 1988

European Journal of Biochemistry

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“…Solutions of flavocytochrome b, were prepared daily from pellets obtained by centrifugation of small aliquots of the stock precipitate, then dissolved in the minimum amount of buffer 11. Flavocytochrome b, preparations had an A,,,(oxidized)/A,,, (reduced) ratio of 0.49 [17]; their molecular activity was determined from standard assays according to [17] and was found to be 1000 t 100 s -' , close to the optimum value. The heme concentrations were measured spectrophotometrically ( F ,~~~ = 183 mM-' .…”

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confidence: 96%

“…A preliminary account of these results has been given [16]. [17]. Solutions of flavocytochrome b, were prepared daily from pellets obtained by centrifugation of small aliquots of the stock precipitate, then dissolved in the minimum amount of buffer 11.…”

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Study of the Hansenula anomala yeast flavocytochrome‐b₂–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

Thomas

Favaudon

François

1983

European Journal of Biochemistry

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Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn‐cytochrome c appears to be in the form of a stable though non‐covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal‐free porphyrin‐cytochrome c, the precursor of Zn‐cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate‐reduced state showed a simple binding equilibrium (Kd=0.1 μM at I=0.03 M, 10°C) with a stoichiometry of one Zn‐cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn‐porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.

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“…The void volume fraction was concentrated using a Centricon microcentrator (Amicon, Danvers, Massachusetts) and stored at -80°C in small aliquots. The specific activity was determined at 30°C by measuring the initial rate of absorbance change at 420 nm of a 20 mM L-lactate and 1 mM ferricyanide solution in 0.1 M phosphate buffer, pH 7.0 (Labeyrie et al, 1978). The concentration of flavocytochrome b2 was determined by measuring the absorbance at 423 nm of the dithionitereduced enzyme, using an extinction coefficient of 183 mM" cm" (Labeyrie et al, 1978).…”

Section: Purification and Asstry Of Flavocytochrome B2mentioning

confidence: 99%

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Rao

Lederer

1998

Protein Science

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Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate a-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on NE of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-'H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, k:, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J BioZ Chem 260: 1 1 1 15-1 1 122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (kO.03) M" s". Using the Eigen equation, this figure yields a pK, of 9.1 f 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, JBiochem 67:291-295). The mechanistic significance of these results is discussed.

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[27] Flavocytochrome b2 or l-lactate cytochrome c reductase from yeast

Cited by 106 publications

References 28 publications

Probing the active site of flavocytochrome b₂ by site‐directed mutagenesis

Probing the active site of flavocytochrome b₂ by site‐directed mutagenesis

Study of the Hansenula anomala yeast flavocytochrome‐b₂–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

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[27] Flavocytochrome b2 or l-lactate cytochrome c reductase from yeast

Cited by 106 publications

References 28 publications

Probing the active site of flavocytochrome b2 by site‐directed mutagenesis

Probing the active site of flavocytochrome b2 by site‐directed mutagenesis

Study of the Hansenula anomala yeast flavocytochrome‐b2–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

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Probing the active site of flavocytochrome b₂ by site‐directed mutagenesis

Probing the active site of flavocytochrome b₂ by site‐directed mutagenesis

Study of the Hansenula anomala yeast flavocytochrome‐b₂–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)