Production and characterization of erythropoietic protoporphyric heterodimeric ferrochelatases

Abstract

Mutations resulting in diminished activity of the dimeric enzyme ferrochelatase are a prerequisite for the inherited disorder erythropoietic protoporphyria (EPP). Patients with clinical EPP have only 10% to 30% of normal levels of ferrochelatase activity, and although many patients with EPP have one mutant allele and one "low-expression" normal allele, the possibility remains that, for some, low ferrochelatase activity may result from an EPP mutation that has an impact on both subunits of the wild-type/mutant heterodimer. Here we present data for 12 ferrochelatase wild-type/EPP mutant heterodimers showing that some mutations result in heterodimers with the residual activity anticipated from individual constituents, whereas others result in heterodimers with significantly lower activity than would be predicted. Although the data do not allow an a priori prediction of heterodimeric residual activity based solely on the in vitro activity of EPP homodimers or the position of the mutated residue within ferrochelatase, mutations that affect the dimer interface or [2Fe-2S] cluster have a significantly greater impact on residual activity than would be predicted. These data suggest that some EPP mutations may result in clinically overt EPP in the absence of a low-expression, wild-type allele; this is of potential significance for genetic counseling of patients with EPP.

Figure 1.

UV-visible spectra for purified wild-type and mutant human ferrochelatases. The key identifies the proteins examined. All other mutant proteins showed similar spectra except for the proteins with the mutations M288K, H386P, C406S, and F417S (see “Results”). WT indicates wild type.

Figure 2.

Active site–located EPP mutations. The catalytically essential H263 (which is not an identified EPP mutation) is shown as a CPK space-filling model with carbon atoms colored violet as a landmark for the central portion of the active-site pocket. EPP active-site mutant residues described in the present study are at positions P334 (carbons in green), Y191 (carbons in light gray), and S264 (carbons in yellow). All structure figures were generated with PyMOL.

Figure 3.

Positions of mutations near the dimer interface and the [2Fe-2S] cluster. Dimer from one side. Residues at the dimer interface are M267 (carbons in orange) and M288 (carbons in violet). Residues in proximity to the [2Fe-2S] cluster are C406 (carbons in yellow) and F417 (carbons in light gray). In this figure and in Figure 4, the [2Fe-2S] cluster is shown in black. One subunit is rendered in green and the other in slate blue.

Figure 4.

Location of other EPP mutations. Single monomeric subunit. The surface presented to the viewer is the backside of the protein that faces toward the mitochondrial matrix and is opposite the active-site pocket face. Residues shown are Q139 (carbons in yellow), K379 (carbons in violet), C236 (carbons in orange), F260 (carbons in light gray), and H386 (carbons in green).