Iron-activated alcohol dehydrogenase from Zymomonas mobilis: isolation of apoenzyme and metal dissociation constants

The title enzyme contains ferrous iron in its native, active form. Inclusion of Co super(2+) in the isolation buffer leads to (0% substitution of Fe super(II) for Co super(II), providing a preparation that is more stable and also active. Treatment of the Co super(II) enzyme with 1,10-phenantroline leads to apoenzyme (< 3 atom % total metal content), which can be completely reactivated upon addition of a single equivalent of Fe super(2+) or Co super(2+). Zn super(2+) is ineffective under the same conditions. Apparent metal dissociation constants, K sub(M), were determined for the bivalent metal ions of the first transition series. That for Co super(II) was defined by using nitrilotriacetic acid as metal buffer, while the remainder were determined by competition of two metal ions for the apoenzyme. Complementary experiments in which the total concentration of first one metal ion and the other was kept constant were employed to maximize precision. The K sub(M) values increase slowly with atomic number and do not follow the classic Irving-Williams series.