Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III: Propionate interactions and porphyrin core deformation

Abstract

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups.These findings let us to thoroughly describe the physiological cpIIIferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV-vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.Andrea Dali, Thomas Gabler, and Federico Sebastiani contributed equally to this study.

Iron insertion into coproporphyrin III‐ferrochelatase complex: Evidence for an intermediate distorted catalytic species

“…The experiment allowed us to verify that Lm CpfC is active in its crystalline state, even though reaction kinetics presumably are slowed down compared to in‐solution approaches (Schmidt, 2020; Schmidt & Saldin, 2014), due to limited mass exchange rate. The overall structures of the “24%” (soaking time: 2 min), “34%” (soaking time: 3 min), and “68%” (soaking time: 4 min) iron occupancy samples are identical to those of Lm CpfC‐cpIII (PDB ID: http://firstglance.jmol.org/fg.htm?mol=8AT8) (Dali et al, 2023) and Lm CpfC‐coproheme (PDB ID: http://firstglance.jmol.org/fg.htm?mol=6SV3) (Hofbauer et al, 2020). In fact, their secondary structural elements are perfectly superimposable: the “24% Fe” structure shows an rmsd‐value of the backbone carbons compared to the Lm CpfC‐cpIII structure of 0.311 Å and 0.328 Å when aligned with the Lm CpfC‐coproheme structure; the “34% Fe” structure shows rmsd‐values of 0.165 Å (compared to Lm CpfC‐cpIII) and 0.186 Å (compared to Lm CpfC‐coproheme); the “68% Fe” structure shows rmsd‐values of 0.164 Å (compared to Lm CpfC‐cpIII) and 0.183 Å (compared to Lm CpfC‐coproheme).…”

“…In regard to the R29-p7 H-bond interaction, it has been already observed that this H-bond is not always present, since in the LmCpfC-cpIII crystal structure the R29 residue shows two possible conformations, in and out (Figure 2b and Dali et al, 2023). In solution, the R29L variant shows wild-type-like characteristics upon coproheme binding, while upon cpIII binding it shares similarities with the R45L variant, where only the H-bond with p6 is removed (Dali et al, 2023;Gabler et al, 2022). In this work, changes in the orientation of the R29 residue are observed along the metalation process.…”

“…Based on the overall spectroscopic differences, we were able to follow the ferrous iron titration, under anaerobic conditions, of the WT LmCpfC-cpIII complex to the formation of the WT LmCpfC-coproheme complex using both UV-vis absorption and RR spectroscopies. Upon progressive addition of small aliquots (0.1 eq) of Fe 2+ to the WT-cpIII complex, the UV-vis spectra (Figure 3 and Figure S2) change from that typical of WT-cpIII (bands at 405, 507, 544, 560, and 611 nm) (black spectrum) to the final WT-coproheme complex spectrum (bands at 397, 497, 527, and 618 nm) (green spectrum) (Dali et al, 2023;Gabler et al, 2022). The result can be interpreted in terms of different equilibrium populations between the WT LmCpfC-cpIII and WT LmCpfC-coproheme species during titration.…”

Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase – unexpected activities of active site variants

“…The reason for the reduced activity of the ppIXbound WT and the complete loss of activity of its key distal variants is very likely to derive from an incorrect substrate binding, as a consequence of the presence of only two propionate substituents in the substrates. In fact, comparison of the structures, functional and spectroscopic studies of WT and mutated CpfCs from Bacillus subtilis in complex with N-MeMP or deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride [15,35] with those of LmCpfC with coproheme and coproporphyrin III [5][6][7], clearly shows that the interaction of propionates at positions 2 and 4 with residues well buried in the protein core is fundamental for correct binding. The complete ferrous iron insertion also observed in this work by cpIbound WT strengthens this conclusion.…”

“…Ferrochelatases are enzymes which insert ferrous iron into a porphyrin macrocycle within heme biosynthesis pathways [4]. However, while ferrochelatases of monoderm and diderm representatives do not significantly differ from each other in their overall subunit structure and fold, they exhibit differences in the porphyrin binding site architecture [5][6][7]. In addition, in some ferrochelatase representatives of both monoderm and diderm bacteria (e.g., in some actinobacterial ferrochelatases or representatives of Bacteroidetes [8]), a [2Fe-2S] cluster of yet unknown function is found.…”