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Crosstalk between metal ions in Bacillus subtilis ferrochelatase

Hansson, Mattias LU ; Lindstam, Mats LU and Hansson, Mats LU (2006) In Journal of Biological Inorganic Chemistry 11(3). p.325-333
Abstract
Ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of Fe2+ into protoporphyrin IX, generating heme. In vitro assays have shown that all characterized ferrochelatases can also incorporate Zn2+ into protoporphyrin IX. Previously Zn2+ has been observed at an inner metal binding site close to the porphyrin binding site. Mg2+, which stimulates Zn2+ insertion by Bacillus subtilis ferrochelatase, has been observed at an outer metal binding site. Exchange of Glu272 to a serine eliminated the stimulative effect of Mg2+. We found that Zn2+ quenched the fluorescence of B. subtilis ferrochelatase and this quenching was used to estimate the metal affinity. Trp230 was identified as the intrinsic... (More)
Ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of Fe2+ into protoporphyrin IX, generating heme. In vitro assays have shown that all characterized ferrochelatases can also incorporate Zn2+ into protoporphyrin IX. Previously Zn2+ has been observed at an inner metal binding site close to the porphyrin binding site. Mg2+, which stimulates Zn2+ insertion by Bacillus subtilis ferrochelatase, has been observed at an outer metal binding site. Exchange of Glu272 to a serine eliminated the stimulative effect of Mg2+. We found that Zn2+ quenched the fluorescence of B. subtilis ferrochelatase and this quenching was used to estimate the metal affinity. Trp230 was identified as the intrinsic fluorophore responsible for the observed quenching pattern. The affinity for Zn2+ could be increased by incubating the ferrochelatase with the transition state analogue N-methyl mesoporphyrin IX, which reflected a close collaborative arrangement between the two substrates in the active site. We also showed that the affinity for Zn2+ was lowered in the presence of Mg2+ and that bound Zn2+ was released upon binding of Mg2+. In the ferrochelatase with a Glu272Ser modification, the interaction between Zn2+ and Mg2+ was abolished. It could thereby be demonstrated that the presence of a metal at one metal binding site affected the metal affinity of another, providing the enzyme with a site that regulates the enzymatic activity. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
metal interaction, porphyrin metallation, N-methyl mesoporphyrin IX, hemH, ferrochelatase
in
Journal of Biological Inorganic Chemistry
volume
11
issue
3
pages
325 - 333
publisher
Springer
external identifiers
  • pmid:16453119
  • wos:000236586000007
  • scopus:33645522083
ISSN
1432-1327
DOI
10.1007/s00775-006-0080-2
language
English
LU publication?
yes
id
fb233e48-9521-43ec-b128-b04b94f270b6 (old id 414445)
date added to LUP
2007-10-03 18:34:49
date last changed
2019-07-09 01:51:23
@article{fb233e48-9521-43ec-b128-b04b94f270b6,
  abstract     = {Ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of Fe2+ into protoporphyrin IX, generating heme. In vitro assays have shown that all characterized ferrochelatases can also incorporate Zn2+ into protoporphyrin IX. Previously Zn2+ has been observed at an inner metal binding site close to the porphyrin binding site. Mg2+, which stimulates Zn2+ insertion by Bacillus subtilis ferrochelatase, has been observed at an outer metal binding site. Exchange of Glu272 to a serine eliminated the stimulative effect of Mg2+. We found that Zn2+ quenched the fluorescence of B. subtilis ferrochelatase and this quenching was used to estimate the metal affinity. Trp230 was identified as the intrinsic fluorophore responsible for the observed quenching pattern. The affinity for Zn2+ could be increased by incubating the ferrochelatase with the transition state analogue N-methyl mesoporphyrin IX, which reflected a close collaborative arrangement between the two substrates in the active site. We also showed that the affinity for Zn2+ was lowered in the presence of Mg2+ and that bound Zn2+ was released upon binding of Mg2+. In the ferrochelatase with a Glu272Ser modification, the interaction between Zn2+ and Mg2+ was abolished. It could thereby be demonstrated that the presence of a metal at one metal binding site affected the metal affinity of another, providing the enzyme with a site that regulates the enzymatic activity.},
  author       = {Hansson, Mattias and Lindstam, Mats and Hansson, Mats},
  issn         = {1432-1327},
  keyword      = {metal interaction,porphyrin metallation,N-methyl mesoporphyrin IX,hemH,ferrochelatase},
  language     = {eng},
  number       = {3},
  pages        = {325--333},
  publisher    = {Springer},
  series       = {Journal of Biological Inorganic Chemistry},
  title        = {Crosstalk between metal ions in Bacillus subtilis ferrochelatase},
  url          = {http://dx.doi.org/10.1007/s00775-006-0080-2},
  volume       = {11},
  year         = {2006},
}