Rapid kinetic studies of partial reactions in the heme free derivative of L-lactate cytochrome c oxidoreductase (flavocytochrome b<sub>2</sub>); the flavodehydrogenase function

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

Section: Characterization Of the H Anomala Ji'avodehydrogenasesupporting

confidence: 65%

Section: Characterization Of the H Anomala Ji'avodehydrogenasesupporting

confidence: 55%

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

Célérier¹,

Risler²,

Schwencke³

et al. 1989

“…Such an increase has already been observed for the hemeless S-flavocytochrome b, [8] and FDH, [21]. As shown by Iwatsubo et al IS], it was not related to a modification of the Kd value for ferricyanide but to the rate constant, k , of the flavin reoxidation step.…”

Section: Structural and Functional Properties Of The Fdh V8 Moleculesupporting

confidence: 52%

“…about 50 times more than (4%. 8 ) . The resulting pellet was dissolved and the solution was equilibrated in phosphateiurea denaturing buffer (cf.…”

Section: Renaturation Of the Fdh8 Fragnzeritmentioning

confidence: 99%

Proteolytic Cleavage of Hansenula anomala Flavocytochrome b₂ into Its Two Functional Domains

Gervais¹,

Risler²,

Corazzin³

1983

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b, into its two functional domains: a cytochrome h, core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1 :< that of intact flavocytochrome b,. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome 6 , which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting thein is cleaved by Staphylococcus aureus V 8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80 "/, that of flavocytochrome b,. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.Our results are discussed in terms of 'gene fusion'.Each chain of yeast flavocytochrome b, has been shown to be folded into three globules: n, E and / I ' joined together in the 'intact' enzyme by two protease-sensitive bridges aa' and cd [I -41 (cf. These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].From a functional point of view, the first cleavage in aa' results in a 96 %loss of activity [I I]; the isolated 'nicked' FDH, entity itself only retains about 1 % of the ferricyanide reductase activity of native flavocytochrome b2 [lo]. These results did not elucidate whether such a low activity was due to the aa' cleavdge itself or to the release of cytochrome b2 core resulting from the second cleavage at cd.This study presents information concerning this question. Despite the hypersensitivity of the aa' zone towards a number of proteases [I2 -141, we succeeded in obtaining a cleavage in the cd zone faster than in the aa' zone. This was achieved with Hansenula anomala flavocytochrome b2 and the highly specific Staphylococcus aureus V 8 protease 1. We describe a method for separating the so-formed 'intact' flavodehydrogenase part which we currently term 'FDH,,', from the other proteolysis products. Steady-state catalytic parameters of this new flavocytochrome h, derivative have been determined and compared with those of the native flavocytochrome b, and its tryptic derivatives. MATERIALS A N D METHODS Hansenula anomala Flavocytochrome b,This was prepared according to [I51 with the minor modifications described in [I I]. It was stored as an ammonium sulfate precipitate (50 % satur...

“…Recently, Iwatsubo et al [5] published the spectrum of a heme-free cytochrome bz derivative obtained after prosthetic group removal from the denatured protein and dialysis in the presence of FMN. The spectrum of the reconstituted derivative does not show the prominent shoulder at 425 nm that we observe.…”

Section: Discussionmentioning

confidence: 99%

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

Lederer

1978

Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillere Blandin et al. Eur. J. Bioclwm. 54, 549-566 (1975)j and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 pM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavoproteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244,[3999][4000][4001][4002][4003][4004][4005][4006] (1 969)].Free reduced flavin is rapidly reoxidized by oxygen, whereas enzyme-bound flavin either keeps or loses this property, according to the function of the protein. This phenomenon provides the basis for the now classical distinction, among flavoproteins, between oxidases and dehydrogenases. It is clear that the protein moiety plays a fundamental role in modulating the reactivity of the bound flavin, but this role is at present poorly understood, in spite of numerous efforts.A few years ago, Massey et al. [l] proposed an interesting correlation between the reactivity of flavoproteins toward oxygen and toward sulfite. It was shown that a number of flavoproteins were bleached by sulfite, which reversibly formed a covalent bond with N-5 of the flavin nucleus [2]; those proteins that reacted were oxidases and formed anionic (red) semiquinones; those that did not react were dehydrogenases and formed neutral (blue) semiquinones. A tentative explanation was presented, based on these and other observations: a positive charge at the active site of oxidases would facilitate sulfite binding and stabilize the anionic semiquinone ;