Studies on cytochrome b562 of Escherichia coli. II. Reconstitution of cytochrome b562 from apoprotein and hemin.

Abstract Apocytochrome b562 reacts with hemin to form holocytochrome b562. The kinetics of holocytochrome formation show that three separate first order reactions can be recognized in the over-all reconstitution reaction. On this basis, it is postulated that the formation of holocytochrome involves a minimum of four reaction steps which can be formulated as follows: [see PDF for equation] The initial reaction represents the formation of the apocytochrome-hemin complex (X1). This initial reaction, which would be second order, is apparently too rapid to be observed in the stop-flow apparatus (l5 msec) and may be diffusion-controlled. The subsequent first order reactions give rise to intermediate complexes (X2 and X3) and finally to holocytochrome b562. The first order rate constants, k1, k2, and k3 for these reactions at pH 7, are 20.9, 2.64, and 0.098 sec-1, respectively. The rate constant (k3) for the rate-limiting step increases at alkaline pH values and decreases under acidic conditions. The rate constants k1 and k2 show opposite behavior. Both k1 and k2 increase under acidic conditions and decrease at alkaline pH values. The energy of activation of the over-all reconstitution reaction is 18.1 kcal per mole. Diazotization and acetylation of apocytochrome b562 produces in each case a modified apoprotein which retains the ability to combine with hemin. The spectrum of the diazotized cytochrome b562-hemin complex, in the oxidized state, is quite different from that of the native holocytochrome; however, the spectra of the two forms in the reduced state are quite similar. Acetylated apocytochrome b562 and hemin form a complex which exhibits two broad absorption bands centering at 390 and 345 mµ. Reduction of the acetylated apocytochrome-hemin complex yields a complex which has an absorption spectrum identical with that of the reduced form of the native cytochrome. Treatment of apocytochrome b562 with monoiodoacetic acid prior to complex formation with hemin has no effect on the reconstitution reaction. Treatment of apocytochrome b562 with iodine prevents the reconstitution reaction.