Dong-Sun Lee*, Eva Flachsová‡, Michaela Bodnárová‡, Borries Demeler†, Pavel Martásek‡ & C. S. Raman*§
*Structural Biology Research Center, Department of Biochemistry & Molecular Biology, University of Texas - Medical School, Houston, TX 77030; ‡Department of Pediatrics, Center of Integrative Genomics, First School of Medicine, Charles University, 121 09 Prague, Czech Republic; and †Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, 78229
Hereditary coproporphyria (HCP) is an autosomal dominant disorder resulting from the half-normal activity of coproporphyrinogen oxidase (CPO), a mitochondrial enzyme catalyzing the antepenultimate step in heme biosynthesis. Although several mutations in the CPO gene have been described, the structural basis for how these alterations diminish enzyme activity is incompletely understood. Moreover, the mechanism by which CPO catalyzes the extraordinary metal- and cofactor-independent oxidative decarboxylation is also unknown. Here, we show that CPO has a novel tertiary topology comprising an unusually flat seven-stranded b-sheet that is sandwiched by a-helices. The quaternary state is defined by a homodimer (KD = 5.3 ± 1.2 mM, DGdiss » 7.0 kcal mol-1) in which one monomer rotates relative to the second by approximately 40º to create an inter-subunit interface in close proximity to the two independent active sites. Consequently, deletion of the highly conserved region encoded by exon six, detected in the CPO gene of a HCP patient, will generate a protein that can neither dimerize nor sustain activity. Furthermore, most of the disease-causing mutations occur in regions that are indispensable for maintaining the structural integrity of CPO. The unexpected finding of citrate at the active site helps to demarcate the residues critical for substrate binding and catalysis. Thus, Arg262 and Arg413 mediate carboxylate recognition; Gly406 and Leu407 entertain non-bonded contacts; His258 and Asp282 constitute a catalytic diad; Ser244, Asn260, and Ser416 facilitate proton abstraction or stabilize an intermediate. A transition metal center is also absent. Based on these findings, we propose a mechanism in which triplet oxygen serves as the immediate electron acceptor and a substrate radical or a carbanion intermediate with substantial radical character participates in catalysis. Together, our results have broad implications for deciphering the mechanistic puzzle of CPO and for understanding structure-function relationships in HCP. [This work is supported by the Pew Charitable Trusts via a Pew Scholar Award (C.S.R.), the Robert A. Welch Foundation (C.S.R., AU-1574), and GAUK 25/04 (M.B. and P.M.)]