skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase

Abstract

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe2+ chelation into protoporphyrin IX. Resonance Raman and W-visible absorbance spectroscopes of wild type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into protoporphyrin by ferrochelatase. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. Addition of free base or metalated porphyrins to wild type ferrochelatase and H207N variant yields a quasi 1:1 complex, possibly a monomeric protein-bound species. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub-stoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive resonance Raman lines and the vinyl vibrational mode. Resonance Raman spectra of free base and metalated porphyrins bound to the wild type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle, although the magnitude of the distortion cannotmore » be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decomposition indicates a homogeneous ruffled distortion for the nickel protoporphyrin bound to the wild type ferrochelatase, whereas both a planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance , 3 Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together these findings suggest that the mode of porphyrin distortion in murine ferrochelatase is different from that reported for yeast ferrochelatase, which requires metal binding for porphyrin distortion.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Sandia National Laboratories (SNL), Albuquerque, NM, and Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
7053
Report Number(s):
SAND99-1253J
ON: DE00007053
DOE Contract Number:  
AC04-94AL85000
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Name: Biochemistry
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; Porphyrins; Chelating Agents; Mice; Mutants

Citation Formats

Ferreira, Gloria C, Franco, Ricardo, Lu, Yi, Ma, Jian-Guo, and Shelnutt, John A. Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase. United States: N. p., 1999. Web.
Ferreira, Gloria C, Franco, Ricardo, Lu, Yi, Ma, Jian-Guo, & Shelnutt, John A. Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase. United States.
Ferreira, Gloria C, Franco, Ricardo, Lu, Yi, Ma, Jian-Guo, and Shelnutt, John A. 1999. "Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase". United States. https://www.osti.gov/servlets/purl/7053.
@article{osti_7053,
title = {Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase},
author = {Ferreira, Gloria C and Franco, Ricardo and Lu, Yi and Ma, Jian-Guo and Shelnutt, John A},
abstractNote = {Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe2+ chelation into protoporphyrin IX. Resonance Raman and W-visible absorbance spectroscopes of wild type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into protoporphyrin by ferrochelatase. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. Addition of free base or metalated porphyrins to wild type ferrochelatase and H207N variant yields a quasi 1:1 complex, possibly a monomeric protein-bound species. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub-stoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive resonance Raman lines and the vinyl vibrational mode. Resonance Raman spectra of free base and metalated porphyrins bound to the wild type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle, although the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decomposition indicates a homogeneous ruffled distortion for the nickel protoporphyrin bound to the wild type ferrochelatase, whereas both a planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance , 3 Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together these findings suggest that the mode of porphyrin distortion in murine ferrochelatase is different from that reported for yeast ferrochelatase, which requires metal binding for porphyrin distortion.},
doi = {},
url = {https://www.osti.gov/biblio/7053}, journal = {Biochemistry},
number = ,
volume = ,
place = {United States},
year = {Wed May 19 00:00:00 EDT 1999},
month = {Wed May 19 00:00:00 EDT 1999}
}