Detection and classification of hyperfine-shifted H-1, H-2, and N-15 resonances of the Rieske ferredoxin component of toluene 4-monooxygenase

T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required f or electron transfer between the oxidoreductase and diiron hydroxylase comp onents of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preli minary H-1 NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1 996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine -shifted H-1 resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperf ine-shifted H-1, H-2, and N-15 signals from T4MOC. Selective deuteration le d to identification of signals from the four Hii atoms of cluster ligands C 45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state , the Curie temperature dependence of the HE protons corresponded to that p redicted from the simple vector spin-coupling model for nuclei associated w ith the localized ferric site. The signal at 25.5 ppm in the H-1 spectrum o f reduced T4MOC was assigned on the basis of selective H-2 labeling to the His H-epsilon 1 atom of one of the cluster ligands (H47 or H67). This assig nment was corroborated by a one bond H-1-C-13 correlation (at 25.39 ppm H-1 and 136.11 ppm C-13) observed in spectra of [U-C-13]T4MOC with a H-1-C-13 coupling constant of similar to 192 Hz, The carbon chemical shift and one b ond coupling constant are those expected for H-1(epsilon 1)-C-13(epsilon 1) in the imidazolium ring of histidine and are inconsistent with values expe cted for cysteine H-1(alpha)-C-13(alpha). The His H-epsilon 1 proton exhibi ted weak Curie temperature dependence from 283 to 303 K, contrary to the an ti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A H-1 peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspo nd to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results wi th T4MOC call into question certain H-1 assignments recently reported on th e basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 1469 0-14696]. Selective N-15 labeling was used to identify hyperfine-shifted N- 15 NMR signals from the backbone nitrogens of ail four cluster ligands (C45 , H47, C64, and H67), from the N-epsilon 2 atoms of the two histidine ligan ds (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) pre sent in the cluster-binding motif of T4MOC in the oxidized and reduced stat es. The results indicate that the N-delta 1 of each of the two ligand histi dines of T4MOC are ligated to an iron atom and reveal a pattern of H-bondin g to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T 4MOC) of the five backbone amide H-bunds expected on the basis of compariso n with the crystal structures of other related Rieske proteins; the fifth b ackbone amide (150 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske protei ns that may be related to functional diversity in this protein family.