Modeling of electrostatic recognition processes in the mammalian mitochondrial steroid hydroxylase system

Adrenodoxin reductase (AR) and adrenodoxin (Adx) are components of the mammalian mitochondrial steroid-hydroxylating system. Crystal structures of Adx, AR and a cross-linked Adx–AR complex have recently been determined. Based on these, we have carried out a modeling and docking study to characterize the recognition between AR, Adx and cytochrome c (Cytc). To rationalize the recognition process, electrostatic potentials were calculated by solving the Poisson–Boltzmann equations. In the Adx–AR complex modeled, a negatively charged surface of Adx recognizes a positive surface of AR, as in the crystal structure of the Adx–AR complex, proving the correct parameterization for the energy calculations. After forming salt bridges between the polar primary binding sites of Adx and AR, charge compensation causes a domain movement in AR, which closes the binding cleft by 2–4 Å. Thereby, a secondary polar binding site is closed and the electron transfer pathways between the FAD of AR and the [2Fe–2S] cluster of Adx are adjusted. Next, the model structure of a complex between Adx and Cytc was derived. The lowest-energy complex between Adx and Cytc matches earlier chemical modification and cross-linking experiments, which proposed polar interactions of Lys13, Lys27, Lys72 and Lys79 of Cytc with acidic residues in Adx. Because of the short distance of 9.4 Å between the redox centers, a complex, productive in electron transfer via a different outlet pathway from the inlet route in Adx, is expected. However, a ternary complex cannot be formed between the Adx–AR complex and Cytc because of steric hindrance. Therefore, a shuttle model for the role of Adx in the electron transfer process to Cytc is preferable to a relay model. In addition, no preferable docking site could be detected for a second Adx when probing the Adx–AR complex, which is required for a quaternary organized-cluster model of all redox partners of the hydroxylase system.