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

Abstract

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

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

“…In the case of cytochrome c, the oxidation steps 4 + 5 and 6 + 9 involving reduced heme (H,) as electron donor have as well been characterized [22]. The oxidation steps 4 + 6 and 5 --+ 9 in which F, is the donor are thought to take place exclusively with ferricyanide [9,[27][28][29][30][31]. The dissociation of pyruvate after lactate oxidation is considered very fast and it does not appear in the scheme.…”

Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli

“…The crystal structure of flavocytochrome b2 has been solved to 0.24 nm resolution [4] and shows that each subunit consists of two distinct domains: an N-terminal cytochrome domain containing protohaem IX and a C-terminal domain containing flavin mononucleotide (FMN). Various attempts have been made to isolate these domains using controlled proteolysis of the intact holoenzyme [5][6][7] and the cytochrome domain has been successfully isolated after trypsin digestion. This domain has more recently been prepared by expression in Escherichia coli, independent of the flavin domain [8].…”

“…In contrast, the flavin domain has proven difficult to isolate using proteolytic methods, particularly because of a highly proteinase-sensitive region within this part of the protein [9]. Celerier et al [7] succeeded in isolating the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis of the intact enzyme by an H. anomala protease preparation.…”

Cellobiose Dehydrogenase

“…Among these, only flavocytochrome b 2 , which dehydrogenates lactate to pyruvate, is well characterized and this enzyme may serve as a model for CDH (13,18,19). Flavocytochrome b 2 is a tetramer; each monomer contains one FMN and one heme b.…”

“…Flavocytochrome b 2 is a tetramer; each monomer contains one FMN and one heme b. The polypeptide chain is organized into two domains, a cytochrome domain to which the heme binds, and a flavodomain to which the FMN binds (13,18). The two domains are linked by a protease-sensitive segment (18).…”