Dissection of porphyrin-induced conformational dynamics in the heme biosynthesis enzyme ferrochelatase.
Human ferrochelatase (EC 4.99.1.1) catalyzes the insertion ferrous iron into protoporphyrin IX as the last step in heme biosynthesis, an essential process to most organisms given the vast intracellular functions of heme. Even with multiple ferrochelatase structures available, the exact mechanism for iron insertion into porphyrin is still a matter for debate. It is clear, however, that conformational dynamics are important for porphyrin substrate binding, initial chelation of iron, insertion of iron into the macrocycle, and release of protoheme IX. In this work we characterize conformational and dynamic changes in ferrochelatase associated with porphyrin binding using the substrate mesoporphyrin (MPIX) and backbone amide hydrogen/deuterium exchange mass spectrometry (HDX-MS). In general, regions surrounding the active site become more ordered from direct or indirect interactions with the porphyrin. Our results indicate that the lower lip of the active site mouth is preorganized for efficient porphyrin binding, with little changes in backbone dynamics. The upper lip region has the most significant change in HDX behavior as it closes the active site. This movement excludes solvent from the porphyrin pocket, but leads to increased solvent access in other areas. A water lined path to the active site was observed, which may be the elusive iron channel with final insertion via the M76/R164/Y165 side of the porphyrin. These results provide a rigorous view of the ferrochelatase mechanism through the inclusion of dynamic information, reveal new structural areas for functional investigation, and offer new insight into a potential iron channel to the active site.
Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487, USA.
Awuri P
AP
Asuru
Mier
M
An
Laura S
LS
Busenlehner
D020134:Catalytic Domain / Q000187:drug effects*; D005294:Ferrochelatase / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000096:biosynthesis*; D006801:Humans; D008652:Mesoporphyrins / Q000378:metabolism / Q000494:pharmacology*; D008958:Models, Molecular; D012997:Solvents / Q000737:chemistry
D008652:Mesoporphyrins; D012997:Solvents; D006418:Heme; D005294:Ferrochelatase