ACTIVE SITE OF HUMAN FERROCHELATASE
H.A. Dailey, C.-K. Wu, W.N. Missaoui, P. Horanyi, B.-C. Wang, J. Rose, and T.A. Dailey
Biomedical and Health Sciences Institute, Department of Microbiology, and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602
Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin to form protoheme IX. In eukaryotes this enzyme is bound to the matrix side of the inner mitochondrial membrane. In animal cells ferrochelatase is a homodimer with monomer molecular weight of approximately 43,000. It possesses a [2Fe-2S] cluster coordinated by four cysteine residues. The crystal structure of human ferrochelatase has previously been determined at 2.0 Ǻ and clearly demonstrated the presence of the active site pocket that is present on a hydrophobic face of the molecule that is proposed to face the mitochondrial membrane
Currently two catalytic models for ferrochelatase exist. One proposes that deprotonation of the macrocycle and iron insertion are catalyzed by a single conserved active site histidine (H263). The second model proposes that deprotonation occurs via the histidine, but that metallation occurs from the opposite side of the active site pocket. Both models suggest that the metallation reaction involves distortion of the porphyrin macrocycle.
Here we have examined the crystal structures of three mutants of human ferrochelatase : H263C, H341C, and F337A. The mutant H263C has no enzyme activity while the other two mutants have significantly decreased activity. Mutation of either H263 or H341 results in the reorientation of M76, R164, H341, E343 and F337 in the active site. However, in the mutant F337A these residues retain their wild-type positions.
We propose that a hydrogen bond network existing among residues H263, H341 and E343, which is disrupted by mutation of any of these residues, is also disrupted during the normal catalytic cycle when H263 abstracts the pyrrolic proton from an incoming porphyrin molecule. Upon protonation of H263, the hydrogen bond between H263 and E343 is broken. This allows E343 and its hydrogen bonded partner, H341, to reorient. As H341 swings to one side, F337, which is normally restrained from movement by the presence of the side chain of H341, swings into the active site pocket. We propose that the resultant movement of F337 causes the distortion of the macrocycle and allows iron insertion.