The Role of Lysyl Residues in the Structure and Reactivity of Cytochrome b5 Reductase

By selective modification of the lysyl residues in cytochrome b5 reductase with ethyl acetimidate, acetic anhydride, succinic anhydride, and trinitrobenzenesulfonate, derivatives were prepared in which one or several of the properties of the native enzyme had been altered. A comparison of these derivatives and the unmodified flavoprotein identified three classes of lysyl residues. The most reactive group includes the residues which are essential for the interaction of the reductase with its natural electron acceptor, cytochrome b5. The remaining lysyl residues, which react in the presence of NADH, are essential for a stable holoenzyme structure and for the normal sequence of apoprotein refolding to an active conformation following acid denaturation. Finally, a single amino group is protected from these reagents by NADH, and appears to be involved in the binding of this substrate and in the direct hydrogen transfer from the pyridine nucleotide to the flavoprotein.