Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal s tep in the heme biosynthetic pathway, the insertion of ferrous iron into pr otoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mito chondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster t hat is coordinated by four Cys residues whose spacing in the primary sequen ce is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe -2S] cluster. Gel filtration chromatography and dynamic light scattering me asurements revealed that the purified recombinant human ferrochelatase in d etergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453 +/- 10 mV for the [2Fe-2S] cluster. The form o f the protein that was crystallized has a single Arg to Leu substitution. T his mutation has no detectable effect on enzyme activity but is critical fo r crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a = 93.5 Angstrom, b = 87.7 Angstrom, and c = 110.2 Angstrom. There are two molecules in the asymmetric unit and the crys tals diffract to better than 2.0 Angstrom resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 Angstrom based upon the Bi jvoet difference Patterson map. (C) 1999 Elsevier Science B.V. All rights r eserved.