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

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 (, ) 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 -strands arranged in the three anti-parallel -sheet topology observed in all Rieske [2Fe-2S] domain proteins. The S of Cys45 and Cys64 and the N¹ of His47 and His67 provide the ligands to the [2Fe-2S] cluster of T4moC. ¹H–¹⁵N HSQC measurements show that both His47-N² and His67-N² 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.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0594-4