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 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 -453+/-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, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.
Burden
A E
AE
Department of Microbiology, Center for Metalloenzyme Studies, University of Georgia, Athens, GA, USA.
Wu
C
C
Dailey
T A
TA
Busch
J L
JL
Dhawan
I K
IK
Rose
J P
JP
Wang
B
B
Dailey
H A
HA
D002850:Chromatography, Gel; D003460:Crystallization; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D005294:Ferrochelatase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D005294:Ferrochelatase