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Structural and physicochemical studies on metal and porphyrin binding by the Bacillus subtilis ferrochelatase

Hansson, Mattias LU (2009)
Abstract
The enzyme ferrochelatase (EC 4.99.1.1) is the terminal enzyme in the biosynthetic pathway of heme b and catalyzes the insertion of Fe(II) into protoporphyrin IX. In this dissertation ferrochelatase from Bacillus subtilis was studied. A new purification method via inclusion bodies was developed that enabled simple purification of recombinant enzyme without tags or modifications that might affect the enzymatic properties. Ferrochelatases in general have the inherent ability to catalyze the incorporation of various divalent metal ions into different porphyrins in vitro. All characterized ferrochelatases can incorporate Zn(II), as well as Fe(II). The first X-ray structure with the substrate metal Fe bound at the active site is presented in... (More)
The enzyme ferrochelatase (EC 4.99.1.1) is the terminal enzyme in the biosynthetic pathway of heme b and catalyzes the insertion of Fe(II) into protoporphyrin IX. In this dissertation ferrochelatase from Bacillus subtilis was studied. A new purification method via inclusion bodies was developed that enabled simple purification of recombinant enzyme without tags or modifications that might affect the enzymatic properties. Ferrochelatases in general have the inherent ability to catalyze the incorporation of various divalent metal ions into different porphyrins in vitro. All characterized ferrochelatases can incorporate Zn(II), as well as Fe(II). The first X-ray structure with the substrate metal Fe bound at the active site is presented in this dissertation. It showed that Fe was coordinated by the same amino acid residues as in a previously observed Zn(II) containing ferrochelatase structure. The role of different amino acid residues in metal binding could hence be extrapolated from Zn(II) affinity estimations that were performed on a series of ferrochelatase variants. His183 and Glu264 were found to be responsible for metal binding. The affinity was determined using fluorescence quenching and the intrinsic flourophore responsive to the observed quenching was identified as Trp230. Fluorescence quenching was also used to directly measure the effect of the alkaline earth metal Mg(II), which previously had been shown to increase activity: it lowered the affinity of the Zn(II) to be inserted by the enzyme. Several other novel X-ray structures of modified ferrochelatases were solved and using time-resolved crystallography the metallation of N-methylmesoporphyrin (N-MeMP), a strong transition-state inhibitor, could be monitored. The specificity of the binding pocket was further validated by co-crystallization of an inactive ferrochelatase variant with another isomeric form of N-MeMP, and the wild-type enzyme with deuteroporphyrin IX 2,4-disulfonic acid, a weak inhibitor. Apart from Fe(II) and Zn(II), B. subtilis ferrochelatase also accelerates the insertion of Cu(II) into porphyrin, in contrast to most other characterized ferrochelatases, which prefer Co(II). In this dissertation a modified ferrochelatase was designed, constructed, and shown to have a reversed metal preference, i.e. the Co(II) activity at steady state had increased at the expense of Cu(II) insertion ability. The structure of this variant was solved and the association of free Cu(II) and Co(II) to the wild-type and the modified ferrochelatase were measured using electron paramagnetic resonance. The results suggested that the apparent divergence in metal specificity between ferrochelatases could be explained by product release rather than the affinity for the metal substrate. (Less)
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author
supervisor
opponent
  • Professor Dailey, Harry, Paul D. Coverdell Center for Biomedical and Health Sciences Institute, University of Georgia, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
metal specificity, metal binding, porphyrin metallation, time resolved crystallography, fluorescence quenching, X-ray structure, inclusion bodies, Bacillus subtilis, heme biosynthesis, ferrochelatase, hemH
pages
136 pages
defense location
Lunds universitet, Kemicentrum, hörsal A
defense date
2009-06-05 10:15
ISBN
978-91-7422-222-7
language
English
LU publication?
yes
id
b3210841-cf08-4a49-83c7-ef84a62bd5af (old id 1393304)
date added to LUP
2009-05-12 13:41:46
date last changed
2016-09-19 08:45:16
@misc{b3210841-cf08-4a49-83c7-ef84a62bd5af,
  abstract     = {The enzyme ferrochelatase (EC 4.99.1.1) is the terminal enzyme in the biosynthetic pathway of heme b and catalyzes the insertion of Fe(II) into protoporphyrin IX. In this dissertation ferrochelatase from Bacillus subtilis was studied. A new purification method via inclusion bodies was developed that enabled simple purification of recombinant enzyme without tags or modifications that might affect the enzymatic properties. Ferrochelatases in general have the inherent ability to catalyze the incorporation of various divalent metal ions into different porphyrins in vitro. All characterized ferrochelatases can incorporate Zn(II), as well as Fe(II). The first X-ray structure with the substrate metal Fe bound at the active site is presented in this dissertation. It showed that Fe was coordinated by the same amino acid residues as in a previously observed Zn(II) containing ferrochelatase structure. The role of different amino acid residues in metal binding could hence be extrapolated from Zn(II) affinity estimations that were performed on a series of ferrochelatase variants. His183 and Glu264 were found to be responsible for metal binding. The affinity was determined using fluorescence quenching and the intrinsic flourophore responsive to the observed quenching was identified as Trp230. Fluorescence quenching was also used to directly measure the effect of the alkaline earth metal Mg(II), which previously had been shown to increase activity: it lowered the affinity of the Zn(II) to be inserted by the enzyme. Several other novel X-ray structures of modified ferrochelatases were solved and using time-resolved crystallography the metallation of N-methylmesoporphyrin (N-MeMP), a strong transition-state inhibitor, could be monitored. The specificity of the binding pocket was further validated by co-crystallization of an inactive ferrochelatase variant with another isomeric form of N-MeMP, and the wild-type enzyme with deuteroporphyrin IX 2,4-disulfonic acid, a weak inhibitor. Apart from Fe(II) and Zn(II), B. subtilis ferrochelatase also accelerates the insertion of Cu(II) into porphyrin, in contrast to most other characterized ferrochelatases, which prefer Co(II). In this dissertation a modified ferrochelatase was designed, constructed, and shown to have a reversed metal preference, i.e. the Co(II) activity at steady state had increased at the expense of Cu(II) insertion ability. The structure of this variant was solved and the association of free Cu(II) and Co(II) to the wild-type and the modified ferrochelatase were measured using electron paramagnetic resonance. The results suggested that the apparent divergence in metal specificity between ferrochelatases could be explained by product release rather than the affinity for the metal substrate.},
  author       = {Hansson, Mattias},
  isbn         = {978-91-7422-222-7},
  keyword      = {metal specificity,metal binding,porphyrin metallation,time resolved crystallography,fluorescence quenching,X-ray structure,inclusion bodies,Bacillus subtilis,heme biosynthesis,ferrochelatase,hemH},
  language     = {eng},
  pages        = {136},
  title        = {Structural and physicochemical studies on metal and porphyrin binding by the Bacillus subtilis ferrochelatase},
  year         = {2009},
}