The metal-binding site of imidazole glycerol phosphate dehydratase; EPR and ENDOR studies of the oxo-vanadyl enzyme |
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Authors: | J Petersen T R Hawkes David J Lowe |
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Institution: | (1) Nitrogen Fixation Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK Fax: +49-30-31421122; e-mail: jp@donor.chem.tu-berlin.de, GB;(2) Zeneca Agrochemicals, Department of Exploratory Plant Sciences, Jealott's Hill Research Station, Bracknell, Berkshire RG12 6EY, UK Fax: +49-1344-413638; e-mail: t.r.hawkes@zeneca.co.uk, GB |
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Abstract: | The apo protein of imidazole glycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae combines stoichiometrically with certain specific divalent metal cations to assemble the catalytically active form comprising
24 protein subunits and tightly bound metal. VO2+ ions react similarly but, uniquely, result in a metallo-protein (VO-IGPD) with neither catalytic activity nor the ability
to bind to the reaction intermediate analogue, 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate. Since VO2+ apparently assembles the quaternary structure correctly, it is used in the present study as a spin probe to investigate the
metal centre coordination environment by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR)
spectroscopy. At neutral pH, the EPR spectrum of VO-IGPD reveals at least three distinct VO2+ sub-spectra with one predominant at low pH. The spin Hamiltonian parameters for some of the sub-spectra are consistent with
51V having nitrogen in the inner-sphere equatorial coordination environment from, most probably, multiple coordinating histidines.
Further evidence for inner-sphere nitrogen ligands is obtained from ENDOR spectroscopy. The spectra of the low rf region show
signals from interactions with 14N which are consistent with couplings to the imino nitrogen of coordinated histidine residues. In addition a number of proton
ENDOR line pairs are resolved. Of the few that disappear upon exchange of the protein into D2O, one most likely originates from the exchangeable proton of the N-H group of a coordinated histidine imidazole. 1H-ENDOR line pairs from non-exchangeable protons with splittings of approximately 3 MHz can be attributed to imidazole carbon
protons. Thus, most of the couplings observed by ENDOR are consistent with being from the imidazole heterocycle of one or
more histidine ligands.
Received: 27 June 1996 / Accepted: 14 March 1997 |
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