Solution structure of T4moC, the Rieske ferredoxin component of the toluene 4-monooxygenase complex

Lars Skjeldal, Francis C. Peterson, Jurgen F. Doreleijers, Luke A. Moe, Jeremie D. Pikus, William M. Westler, John L. Markley, Brian F. Volkman, Brian G. Fox

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11 Scopus citations


Toluene 4-monooxygenase, a four-protein complex from Pseudomonas mendocina KR1, catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The solution structure of the 112-amino-acid Rieske ferredoxin component, T4moC, was determined from 2D and 3D 1H, 13C, and 15N NMR data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space with input from 1650 experimental restraints, including 1264 inter-proton distance restraints obtained from NOEs, 247 non-redundant intra-residue NOEs, 26 hydrogen bond restraints, and 113 dihedral angle (φ, ψ) restraints. The 20 calculated conformers that best satisfied the input restraints were submitted to refinement in explicit solvent to improve the stereochemical quality. With exclusion of ill-defined N- and C-terminal segments (Ser2; His111-Ser112) and residues near to the [2Fe-2S] cluster, the atomic root mean square deviation for the 20 conformers with respect to the mean coordinates was 1.09 Å for the backbone and 1.60 Å for all non-hydrogen atoms. The T4moC structure consists of 10 β-strands arranged in the three anti-parallel β-sheet topology observed in all Rieske [2Fe-2S] domain proteins. The S γ of Cys45 and Cys64 and the Nδ1 of His47 and His67 provide the ligands to the [2Fe-2S] cluster of T4moC. 1H- 15N HSQC measurements show that both His47-Nε2 and His67-Nε2 are protonated at the pH of the NMR experiments. Comparisons are made between the present NMR structure, previous paramagnetic NMR studies of T4moC, and the X-ray structures of other members of the Rieske protein family.

Original languageEnglish
Pages (from-to)945-953
Number of pages9
JournalJournal of Biological Inorganic Chemistry
Issue number8
StatePublished - Dec 2004

Bibliographical note

Funding Information:
Acknowledgements We thank Dr F. Abildgaard (NMRFAM) for assistance in acquiring and processing the NMR data, Dr M. Billeter (Gothenburg University) for help with construction of the HIF definition, and Dr C. Luchinat (University of Florence) for useful discussions on the construction of the Rieske cluster definitions. This work was supported by the National Science Foundation to B.G.F. (Early Career Development Program MCB-9733734, MCB-0316232), NIH grants to J.L.M. (R01 GM 58667, P50 GM64598, P41 LM05799), and the Norwegian Science Foundation to L.S. (NT 142586/432). NMR data were collected at the National Magnetic Resonance Facility at Madison (NMR-FAM), which is supported by a grant from the NIH Biomedical Research Technology Program (P41 RR 02301) and contains instrumentation purchased with funds from P41 RR 02301, the University of Wisconsin, the NSF Biological Instrumentation Program (DMB-8415048), the NIH Shared Instrumentation Program (RR 02781), and the U.S. Department of Agriculture.


  • Iron-sulfur cluster
  • Molecular dynamics
  • Nuclear magnetic resonance
  • Rieske proteins
  • Structure-function relationship

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry


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