Structural consequences of effector protein complex formation in a diiron hydroxylase

Abstract

Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 A from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.

Fig. 1.

Structures of T4moH (gray) and T4moHD aligned by the protomer consisting of TmoA (55 kDa, blue), TmoB (10 kDa, red), and TmoE (35 kDa, green). T4moD (12 kDa) is shown in orange. (A) Overlay of protomers of T4moH and T4moHD. (B and C) Two views of resting T4moHD, with 1 protomer represented as a surface.

Fig. 2.

Stereo images of the T4moH active site. Active-site residues and waters are labeled. Thr-201 is represented as yellow sticks, Asn-202 is represented as green sticks, and Gln-228 is represented as purple sticks throughout this work. (A) Resting T4moH. (B) Resting T4moHD. (C) Sodium dithionite-reduced T4moHD. In all diiron centers described, Glu-134 provides a bidentate bridge between the iron atoms. Additionally, Fe1 is ligated by Glu-104, His-137 ND1, and by HOH3 that is also hydrogen-bonded to Glu-104. Fe2 is ligated by Glu-197 and His-234 NE2. Glu-231 changes conformation in each structure, with the 2 resting state structures showing different monodentate coordination, while the reduced complex has a bidentate, bridging coordination. HOH4 is within hydrogen-bonding distance of the metal-coordinated carboxylates of Glu-134 and Glu-197. Distances and other images are shown in Table S2 and Fig. S1.

Fig. 3.

Changes in the TmoA chain caused by complex formation with T4moD. The TmoA chains in resting T4moH (gray) and the T4moHD complex (blue) are overlaid. The TmoA helices and other residues described in the text are labeled.

Fig. 4.

Channels and cavities found in T4moH are shown as dark surfaces. The lower panels are obtained by an ≈90° rotation from the upper panels. (A) Resting T4moH protomer showing the location of 3 channels from the exterior to the active site (orange mesh). A white star indicates a side pocket to the active site, bounded by Thr-201 (yellow sticks), Asn-202 (green sticks), Gln-228 (purple sticks), and other residues (data not shown). (B) Resting T4moHD showing residues involved in collapse of the outer part of the active-site channel (magenta mesh, Trp-89, Gln-204, Gly-207, Leu-208, Ala-210, Asp-211, Glu-214, Ala-214, Thr-281, Pro-282, Asp-285, Ser-287, Gln-288, and Phe-293), Thr-201 (yellow sticks), Asn-202 (green sticks), and Gln-228 (purple sticks). Asn-202 and Gln-228 have moved into the side pocket of 4A upon complex formation. (C) Sodium dithionite-reduced T4moHD. The comparable surface in resting T4moHD (blue mesh) is also shown. Minor changes localized near to Glu-197 and to Glu-231 were observed upon reduction.