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

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Abstract
Flavocytochrome b2 from baker''s yeast is a bifunctional tetrameric protein which carries 2 prosthetic groups, FMN and heme, per subunit of MW 58,000. The amino terminus of the subunit is wrapped around the heme and constitutes the so-called cytochrome b2 core (MW 11,000), homologous to cytochrome b5. 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. The conditions for selective proteolysis of intact flavocytochrome b2 and of its various previously studied stable nicked forms by the protease from Staphylococcus aureus V8 are described. Successive attack by a combination of 2 proteases is also described. The amino acid sequence of the area where proteolytic attack takes places was established. Chymotrypsin and S. aureus protease open only 1 bond, whereas yeast proteases remove 5 residues from the central part. The various nicked forms, some of which have lost up to 16 amino acid residues, were enzymatically characterized. The flavodehydrogenase part of flavocytochrome b2 may be composed of 2 domains, linked by the region accessible to proteases. That area might constitute a hinge or a clasp between the domains.