The Characterization and Interconversions of Two Conformational States of Cytochrome b5 Reductase

Two stable conformational states of cytochrome b5 reductase, the active holoenzyme and an inactive flavoprotein species, have been obtained in identical buffers between pH 10.7 and 11.5. Physical and chemical characterization of these two protein forms showed that the catalytically active species is a much more compact polypeptide in which one or both of the tryptophanyl residues and the flavin are in a more nonpolar environment. The transition from holoenzyme to the more swollen enzyme form also exposes one cysteinyl and one tyrosyl residue to the medium and destroys the pyridine nucleotide-binding site. Moreover, the swollen enzyme conformation, in contrast to the active enzyme, is attacked by proteolytic enzymes. Prolonged tryptic digestion produced a compact, stable flavin-binding peptide of approximately 10,000 molecular weight. The characterization of the pH dependence and kinetics of the transitions between the two reductase structures suggests that the state of ionization of basic amino acid residues, as well as exposure of the buried cysteinyl and sulfhydryl residues, contribute significantly to the stability of the two species in the alkaline pH region and the observed hysteresis during the cycle of inactivate enzyme formation and regeneration of active reductase. The swollen, inactive conformation is stabilized between pH 8.5 and 10.3 by formation of the mersalyl complex with the additional sulfhydryl group which is reactive in this protein species.