Evidence by NMR for mobility of the cytochrome domain within flavocytochrome b2

Labeyrie, Françoise; Belœil, Jean‐Claude; Thomas, Marie‐Antoinette

doi:10.1016/0167-4838(88)90018-0

Cited by 35 publications

(28 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The low affinity between the domains (Kd > 10 pM) suggests that there is little interaction between them in the native enzyme. This is in line with crystallographic [33] and NMR [34] data which suggest that in the whole uncleaved enzyme the cytochrome b2 core may move relative to the flavodehydrogenase moiety. However their separation may result not only from a single-point cleavage but also from the subsequent loss of peptides.…”

Section: Separation Of the ~W Osupporting

confidence: 69%

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b₂ after mild proteolysis by an H. anomala proteinase

Célérier¹,

Risler²,

Schwencke³

et al. 1989

European Journal of Biochemistry

View full text Add to dashboard Cite

Laboratoire associe a I'Universite Pierre et Marie Curie, Gif-sur-Yvette The protomeric chain of Hansenula anomala flavocytochrome bz was previously shown to be built as the covalent association of two functional domains : an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H . anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer ( M , = 4 x 39000) containing FMN as expected and not heme. It has high L-1actate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same K,,, for L(+)-lactate as flavocytochrome hz, but it has no L-lactate: cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochromc hz. The subcellular origin of the H . anomala proteinase in the preparation has not yet been elucidated.Yeast flavocytochrome b, is a mitochondria1 enzyme located in the interinembrane space [I] and is involved in an electron pathway alternative to the main respiratory chain [2]. It was crystallized more than 30 years ago by Appleby and Morton [3] and has emerged as a model enzyme for the understanding of electron transfer either between a flavoprotein and a cytochrome or between two cytochromes (references in [4]). The mature enzyme is a tetramer [S] ( M , = 240000) 15 -81. Each protomer, in the native state, consists of a single polypeptide chain [7, 91 and

show abstract

Section: Separation Of the ~W Osupporting

confidence: 69%

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b₂ after mild proteolysis by an H. anomala proteinase

Célérier¹,

Risler²,

Schwencke³

et al. 1989

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…In the crystal structure, 1 subunit (S2) out of the two observed in the asymmetric unit gives evidence for mobility of the hemebinding domain relative to the flavodehydrogenase. NMR experiments suggest this mobility persists in solution (Labeyrie et al, 1988). It could be that in solution the heme-binding domain spends very little time docked to its redox partner, just enough for electron transfer to occur during turnover whenever the flavin happens to be reduced.…”

Section: Discussionmentioning

confidence: 99%

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Balme

Lederer

1994

Protein Science

View full text Add to dashboard Cite

The family of FMN-dependent, a-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate a-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins &flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, , J Biol Chem 260: 11 115-1 1122). This suggested the occurrence of a pK, increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and 0 4 , a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pK, drop of about half a p H unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pK, increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.

show abstract

“…Actually, independent evidence for such a mobility was obtained by NMR studies [41] on the oxidized enzyme in solution. In this subunit, a molecule of ligand, recently identified as pyruvate [42], lies parallel to the plane of the isoalloxazine ring at a distance of nearly 0.4 nm [4, 51.…”

Section: Pyruvate Binding and Redox Equilibriamentioning

confidence: 98%

Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b₂) by product binding to the semiquinone transient

Tegoni

Janot

Labeyrie

1990

European Journal of Biochemistry

View full text Add to dashboard Cite

Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (F,) to the cytochrome h2 moiety of flavocytochrome h2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39-45] and to stabilize markedly the F, state of the prosthetic flavin, relative to the oxidized (F,) and the reduced (Fh) states [Tegoni, M., Janot, J.-M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the F,-pyruvate complex is actually much more stable than the F,-pyruvate and Fh-pyruvate complexes.The inhibition produced by pyruvate has been characterized under steady-state conditions using both ferricytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to F, and an uncompetitive effect due to binding to the F, intermediate in a dead-end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.Several years ago, we observed that pyruvate, the product of the oxidation of L-lactate in the presence of Hunsenulu anomnlu L-lactate : cytochrome-c reductase (flavocytochrome b2), has a dramatic effect on the enzyme, an effect never before suspected. Indeed, we have shown that it markedly modifies the two mid-point potentials of the prosthetic flavin, with stabilization of the semiquinone [l, 21, and alters the rate of the intramolecular electron transfer between flavin and heme The present study has becn carried out in order to reexamine the interaction between pyruvate and the different redox forms of the prosthetic flavin and to understand in detail the action of pyruvate on the mechanism of the enzyme. This system has a particular interest since the three-dimensional structure of the homologous enzyme from Succhuromyces cerevisiue has been solved at 0.24-nm resolution [4, 51. In the structure, one molecule of pyruvate is actually detected at the active site, approximately parallel to the isoalloxazine group of the flavin, at a distance of about 0.4 nm from the N(5).Up to now. the kinetic behaviour of flavocytochrome b2 has been interpreted in terms of a reaction scheme established by Capeillirre-Blandin et al. on the basis of stopped-flow and rapid-freezing studies of the partial reactions, study of the 131. The present results now provide a precise understanding of the steps modified in the scheme when pyruvate is present ...

show abstract

Evidence by NMR for mobility of the cytochrome domain within flavocytochrome b2

Cited by 35 publications

References 14 publications

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b₂ after mild proteolysis by an H. anomala proteinase

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b₂ after mild proteolysis by an H. anomala proteinase

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b₂) by product binding to the semiquinone transient

Contact Info

Product

Resources

About

Evidence by NMR for mobility of the cytochrome domain within flavocytochrome b2

Cited by 35 publications

References 14 publications

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b2) by product binding to the semiquinone transient

Contact Info

Product

Resources

About

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b₂ after mild proteolysis by an H. anomala proteinase

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b₂ after mild proteolysis by an H. anomala proteinase

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b₂) by product binding to the semiquinone transient