Porphyrin binding and distortion and substrate specificity in the ferrochelatase reaction: the role of active site residues

Abstract

The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation.

Fig. 1

A representation of deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP) and the N-methylmesoporphyrin (N-MeMP) isomer structures. (I) The (R)-enantiomer of N_A, which was bound to wild-type B. subtilis ferrochelatase. (II) The (S)-enantiomer of N_B, which was bound to the His183Ala variant. (III) dSDP.

Fig. 2

Binding of N-MeMP to the His183Ala variant of ferrochelatase. (a) A stereo view showing N-MeMP regio-isomer N_B in the active site of the His183Ala variant ferrochelatase. (b) The electron density of N-MeMP calculated with coefficients (3F_obs−2F_calc) superimposed on the structure of the inhibitor. All figures were prepared using PyMOL [http://pymol.sourceforge.net/].

Fig. 3

NSD of the X-ray crystallographic structures of N-MeMP and mesoporphyrin bound to ferrochelatase and a catalytic antibody. Distortion of N-MeMP in solution is shown for comparison.

Fig. 4

Binding of dSDP to wild-type ferrochelatase. (a) A stereo view showing the interactions of dSDP with ferrochelatase. (b) Superposition of the structures of ferrochelatase in complex with N-MeMP (yellow) and dSDP (green), showing the relative position of the two inhibitors within the porphyrin-binding cleft. (c) The electron density of dSDP calculated with coefficients (3F_obs−2F_calc) superimposed on the structure of the inhibitors.

Fig. 5

Comparison of ferrochelatase structures. (a) Superposition of the S. cerevisiae ferrochelatase structure (in yellow; PDB code 1LBQ) on the porphyrin-free (green; PDB code 2HRC) and porphyrin complex (magenta; PDB code 2HRE) of Arg115Leu and Glu343Lys variants, respectively, of human ferrochelatase. The porphyrin molecule (blue) is shown with sticks. (b and c) Superposition of the B. subtilis ferrochelatase structure in complex with N-MeMP (in gray, with helices 1 and 2 highlighted in red; PDB code 1C1H) on the yeast ferrochelatase monomers with closed (b) and open (c) conformations. N-MeMP (green) is shown with sticks. The steric clash of the propionic acid side-chains of N-MeMP with the protein structure is visible in b.