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The rieske-type iron-sulfur cluster in 2,4,5-T monooxygenase from Pseudomonas cepacia AC1100
538 Abstracts
J19
T H E R I E S K E - T Y P E I R O N - S U L F U R C L U S T E R IN 2,4,5-T MONOOXYGENASE FROM PSEUDOMONAS CEPACIA A C l l 0 0 M.K. Bowman', Luying Xun a'b, R.M. Davydov a'c, and S.A. Dikanov ~'d
"Macromolecular Structure and Dynamics, Environmental Molecular Sciences Laboratory, Pacific Northwest Laboratory, Richland WA 99352, U.S.A.; hDepartment of Microbiology, Washington State University at Tri-Cities, Richland WA 99352, U.S.A.; Clnstitute of Chemical Physics, Moscow 117977, Russia; dlnstitute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia Pseudomonas cepacia A C l l 0 0 is able to use the chlorinated compound 2,4,5trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. The initial step in the biodegradation of 2,4,5-T by P. cepacia AC1100 is its conversion to 2,4,5-trichlorophenol (2,4,5-TCP). A DNA region from the A C l l 0 0 genome subcloned as a 2.7-kb SstI-XbaI D N A fragment allows the conversion of 2,4,5-T to 2,4,5-TCP on transfer to Pseudomonas aeruginosa PAO1. This 2.7-kb DNA fragment encodes two polypeptides designated TftA1 and TftA2 with molecular masses 51 and 18-kDa, respectively. Elemental analysis of TftA1 and TftA2 indicate that they contain three Fe and two S atoms, strongly suggesting the presence of an [2Fe-2S] cluster. Analysis of the deduced amino acid sequences of TftA1 and TftA2 proteins show homology to the c~- and 13-subunits of multicomponent aromatic dioxygenases and this complex does, in fact, have monooxygenase activity. TftA1 has two conserved cysteine-hystidine pairs at the identical positions as in the other ~-subunits. This motif is also repeated in Rieske iron-sulfur proteins. CW EPR and 1D- and 2D-ESEEM spectroscopy studies were performed with this iron-sulfur cluster in TftA 1 and TftA2 proteins reduced both chemically and by lowtemperature (77 K) y-irradiation. EPR spectra of the chemically reduced cluster show typical rhombic g-tensor symmetry with principal values g,=1.76, g2=1.9 I, g3=2.01. ESEEM spectra of this sample contain two pairs of double-quantum transitions, one with 2.5 and 6.1 MHz and the other 3.6 and 7.7 MHz from two nitrogens with isotropic hyperfine couplings 3.4 and 5.1 MHz. These data indicate a Rieske type [2Fe-2S] cluster where two histidine residues coordinate one iron. The g-tensor and ESEEM frequencies of the chemically reduced cluster coincide with those of the cluster reduced at low temperature indicating that reduction produces negligible structural perturbation. This work provides additional support for the widely conserved structure of Rieske center in different proteins and its high structural stability in different oxidation states. Pacific Northwest Laboratory is a multiprogram national laboratory operated by Battelle Memorial Institute for the U.S. Department of Energy under contract DE-AC06-76RLO 1830. This research was supported by Associated Western Universities, Inc., Northwest Division (AWU NW) under Grant DE-FG06-89ER-75522 or DE-FG06-92RL-12451 with the U.S. Department of Energy.
J19
T H E R I E S K E - T Y P E I R O N - S U L F U R C L U S T E R IN 2,4,5-T MONOOXYGENASE FROM PSEUDOMONAS CEPACIA A C l l 0 0 M.K. Bowman', Luying Xun a'b, R.M. Davydov a'c, and S.A. Dikanov ~'d
"Macromolecular Structure and Dynamics, Environmental Molecular Sciences Laboratory, Pacific Northwest Laboratory, Richland WA 99352, U.S.A.; hDepartment of Microbiology, Washington State University at Tri-Cities, Richland WA 99352, U.S.A.; Clnstitute of Chemical Physics, Moscow 117977, Russia; dlnstitute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia Pseudomonas cepacia A C l l 0 0 is able to use the chlorinated compound 2,4,5trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. The initial step in the biodegradation of 2,4,5-T by P. cepacia AC1100 is its conversion to 2,4,5-trichlorophenol (2,4,5-TCP). A DNA region from the A C l l 0 0 genome subcloned as a 2.7-kb SstI-XbaI D N A fragment allows the conversion of 2,4,5-T to 2,4,5-TCP on transfer to Pseudomonas aeruginosa PAO1. This 2.7-kb DNA fragment encodes two polypeptides designated TftA1 and TftA2 with molecular masses 51 and 18-kDa, respectively. Elemental analysis of TftA1 and TftA2 indicate that they contain three Fe and two S atoms, strongly suggesting the presence of an [2Fe-2S] cluster. Analysis of the deduced amino acid sequences of TftA1 and TftA2 proteins show homology to the c~- and 13-subunits of multicomponent aromatic dioxygenases and this complex does, in fact, have monooxygenase activity. TftA1 has two conserved cysteine-hystidine pairs at the identical positions as in the other ~-subunits. This motif is also repeated in Rieske iron-sulfur proteins. CW EPR and 1D- and 2D-ESEEM spectroscopy studies were performed with this iron-sulfur cluster in TftA 1 and TftA2 proteins reduced both chemically and by lowtemperature (77 K) y-irradiation. EPR spectra of the chemically reduced cluster show typical rhombic g-tensor symmetry with principal values g,=1.76, g2=1.9 I, g3=2.01. ESEEM spectra of this sample contain two pairs of double-quantum transitions, one with 2.5 and 6.1 MHz and the other 3.6 and 7.7 MHz from two nitrogens with isotropic hyperfine couplings 3.4 and 5.1 MHz. These data indicate a Rieske type [2Fe-2S] cluster where two histidine residues coordinate one iron. The g-tensor and ESEEM frequencies of the chemically reduced cluster coincide with those of the cluster reduced at low temperature indicating that reduction produces negligible structural perturbation. This work provides additional support for the widely conserved structure of Rieske center in different proteins and its high structural stability in different oxidation states. Pacific Northwest Laboratory is a multiprogram national laboratory operated by Battelle Memorial Institute for the U.S. Department of Energy under contract DE-AC06-76RLO 1830. This research was supported by Associated Western Universities, Inc., Northwest Division (AWU NW) under Grant DE-FG06-89ER-75522 or DE-FG06-92RL-12451 with the U.S. Department of Energy.