Electron-nuclear double resonance spectroscopy of sup 15 N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the ( sup 2 Fe-2S) Rieske-type clusters

Journal Article · Tue May 30 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00437a051· OSTI ID:5413168

Gurbiel, R J; Batie, C J; Sivaraja, M; True, A E; Fee, J A; Hoffman, B M; Ballou, D P ^[1]

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1. Northwestern Univ., Evanston, IL (USA)

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type (2Fe-2S) cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) (15N)histidine in a 14N background; (4) (14N)histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske (2Fe-2S) center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type (2Fe-2S) centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Moessbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled (Fe2+ (S = 2), Fe3+ (S = 5/2)) pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.

• OSTI ID: 5413168
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:11; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AZOLES
BACTERIA
CARBOXYLIC ACIDS
COMPARATIVE EVALUATIONS
ELECTRONS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
IMIDAZOLES
ISOTOPE APPLICATIONS
ISOTOPES
LEPTONS
LIGANDS
LIGHT NUCLEI
MAGNETIC RESONANCE
METALLOPROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN 14
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
OXYGENASES
PROTEINS
PSEUDOMONAS
RAMAN SPECTRA
RESONANCE
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES