Study of a Zone Highly Sensitive to Proteases in Flavocytochrome b2 from Saccharomyces cerevisiae

Abstract

Flavocytochrome bz from baker's yeast is a bifunctional tetrameric protein which carries two prosthetic groups, FMN and heme, per subunit of M, 58000. The amino terminus of the subunit is wrapped around the heme and constitutes the so-called cytochrome bz core (Mr 11 000), homologous to cytochrome bs. It has been shown in the past that a number of proteases (yeast proteases, chymotrypsin) preferentially cleave the peptide chain at a point situated much further down the polypeptide chain than the C terminus of the heme-binding domain. Some enzymatic parameters are concomitantly modified, but not the quaternary structure. This paper describes the conditions for selective proteolysis of intact flavocytochrome bz and of its various previously studied stable nicked forms by the protease from Staphylococcus aureus V8. Successive attack by a combination of two proteases is also described. We have established the amino acid sequence of the area where proteolytic attack takes places, and shown that chymotrypsin and S. aureus protease open only one bond, whereas yeast proteases remove five residues from the central part. The various nicked forms, some of which have lost up to 16 amino acid residues, have been enzymatically characterized.These and previous results lend support to, but do not prove, the idea that the flavodehydrogenase part of flavocytochrome h2 may be composed of two domains, linked by the region accessible to proteases. That area might constitute a hinge or rather a clasp between the domains.

Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

“…The fragment designated 0' was resistant to high levels of trypsin and was used to quantify assembly of cytochrome b2. Generation of 0' involves cleavage in a proteasesensitive loop within the flavin domain (Ghrir & Lederer, 1981;Xia & Mathews, 1990). Assuming that all of the lysine residues in this loop are good substrates for trypsin, 0' should contain three of the nine methionine residues in mature cytochrome b2.…”

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N

“…5 ) always leads to an increase in the substrate K,,, value and a decrease in the kc,, value (Ghrir & Lederer, 1981). It was noted that this is a surprising situation because one would not expect alterations in a flexible region to entail functional changes, indicative of the existence of some interactions.…”

“…7). After electrotransfer to an Immobilon membrane, amino acid sequencing of the P fragment showed cleavage to have taken place after M307, as for the wild-type enzyme (Ghrir & Lederer, 1981). In contrast, the flavin-free form showed a more complicated cleavage pattern.…”

“…Single cleavages in the same area can be obtained with defined proteases under controlled conditions ( Fig. 5 ) (Pompon & Lederer, 1976;Ghrir & Lederer, 1981). Interestingly, this protease-sensitive region corresponds more or less in the three-dimensional structure to a disordered segment (residues 300-316) that is part of a polypeptide excursion between barrel strand p 4 and helix a 4 (Xia & Mathews, 1990 …”

“…Cleavage points by proteases along the peptide chain of wild-type and mutant flavocytochrome b2. Upper line shows cleavage points of the wild-type polypeptide as determined in a previous study (Ghrir & Lederer, 1981). Bottom line shows cleavage points determined in this study for the Y254L mutant (the two forms).…”