Metal substitutions at the diiron sites of hemerythrin and myohemerythrin: contributions of divalent metals to stability of a four-helix bundle protein.

A general method is described for substitution of Mn(II) and Co(II) into the diiron sites of hemerythrin and myohemerythrin. Characterizations of these metal-substituted proteins show that their structures closely resemble those of the native proteins. In particular, the four-helix bundle structure appears to be maintained. The apomyohemerythrin retains most of the native helix content but is considerably less stable to denaturation than are the metal-containing proteins. The relative affinities of M(II) for apohemerythrin--namely, Co greater than Fe greater than Mn--parallel the stabilities of the M2myohemerythrins to denaturation by guanidinium chloride. These results indicate that for myohemerythrin (i) the majority of the helical structure found in the native protein does not require incorporation of M(II) and (ii) stabilization of the native structure relative to the fully unfolded structure appears to be due predominantly to M(II)-protein interactions, at least for M = Fe and Co. Incorporation of M(II) also generates unfolding cooperativity in myohemerythrin. This cooperativity can be attributed to interhelical interactions, which are prevented in the apoprotein by solvation of the seven metal ligand residues. The results are consistent with a minimal model for folding/unfolding of myohemerythrin and hemerythrin subunits consisting of the sequential equilibria, N in equilibrium with I in equilibrium with D, between native, intermediate, and fully unfolded states, respectively. The properties of apomyohemerythrin make it a candidate for the intermediate state, I.