Binding of Protoporphyrin IX and Metal Derivatives to the Active Site of Wild-Type Mouse Ferrochelatase at Low Porphyrin-to-Protein Ratios

Abstract

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe(2+) chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.

Chelatases: distort to select?

“…The main calculated contributions to the distortion of N-MeMP and Cu-N-MeMP bound to B. subtilis ferrochelatase are saddling, followed by x-waving, ruffling and doming [27]. We reiterate that saddling is an out-of-plane deformation that exposes both the protons and the lone pairs of the nitrogen atoms of the porphyrin macrocycle, in keeping with a porphyrin arrangement for metal insertion.…”

“…The frequency shifts in the structuresensitive lines of the bound porphyrins relative to the unbound porphyrins reflect the type of distortion that is undergone by the porphyrin macrocycle. Significantly, γ 15 , a saddlingsymmetry (B 2u ) out-of-plane mode, emerges with the binding of free-base protoporphyrin to murine ferrochelatase [27] and to the catalytic antibody [21]. A saddling deformation has also been identified as a chief component of the nonplanar distortion of either N-MeMP or Cu-N-MeMP bound to B. subtilis ferrochelatase in the crystal structures of these complexes [20].…”

“…Porphyrin binding and distortion in Saccharomyces cerevisiae and murine ferrochelatases and in antibody 7G12 have been directly assessed using resonance Raman spectroscopy [21,[25][26][27]. In both enzymes and the antibody, porphyrin binding activates several assigned out-of-plane vibrational modes of the macrocycle.…”

“…In fact, the normal-coordinate structural decomposition of the porphyrins contained in the crystal structures accounts for how the different symmetric out-of-plane deformations (ruffled, saddled, domed, x-and y-waved, and propellered structures) contribute to the porphyrin distortion [27]. The main calculated contributions to the distortion of N-MeMP and Cu-N-MeMP bound to B. subtilis ferrochelatase are saddling, followed by x-waving, ruffling and doming [27].…”

“…Independent experimental approaches have provided unequivocal evidence that porphyrin binding to ferrochelatase induces distortion of porphyrin [20,27]. Although axial ligation is not crucial for porphyrin distortion in the active site of ferrochelatase [20,27], however, the catalytic antibody [19] and probably Met8p [24] use axial ligation to induce distortion in the tetrapyrrole macrocycle.…”