Short crystallization paper Human ferrochelatase : crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer Amy E. Burden a , Chia-Kuei Wu b , Tamara A. Dailey b , Johanneke L.H. Busch a , Ish K. Dhawan c , John P. Rose b , Bi-Cheng Wang b , Harry A. Dailey a;b; * a Department of Microbiology, Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-7229, USA b Department of Biochemistry and Molecular Biology, Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-7229, USA c Department of Chemistry, Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-7229, USA Received 20 July 1999; received in revised form 24 August 1999; accepted 2 September 1999 Abstract Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence 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 measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of 3453 þ 10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2 1 2 1 2 1 and have unit cell constants of a = 93.5 A î , b = 87.7 A î , and c = 110.2 A î . There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A î resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A î based upon the Bijvoet difference Patterson map. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Ferrochelatase ; [2Fe-2S] cluster ; Protein crystallography ; Protein expression The enzyme ferrochelatase (EC 4.99.1.1) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Over two dozen DNA sequences for ferrochelatase exist in GenBank although no Archael ferrochelatases have been re- ported. The animal [1^4], but not plant or bacterial, ferrochelatases have been reported to possess a [2Fe- 2S] cluster. While it is known that this cluster is sensitive to destruction by nitric oxide (NO) [5], its speci¢c role in structure stabilization or catalysis is at present unknown. With the exception of the enzymes from Bacillus subtilis [6] and Azospirillum brasilense [7], ferrochelatase has been shown to utilize only the divalent cations of iron, zinc, and cobalt as metal substrates [8]. Trivalent and monovalent metal ions have no e¡ect on enzyme activity and are not sub- 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00196-X * Corresponding author. Fax: (706) 5427567; E-mail : hdailey@arches.uga.edu Biochimica et Biophysica Acta 1435 (1999) 191^197 www.elsevier.com/locate/bba