Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan‐modified NADPH‐adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH‐adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron‐transport chain that transfers electrons from NADPH to cytochromes P‐450 through FAD in AR and [2Fe‐2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N‐bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS‐modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe‐2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition. Proteins 2004. © 2004 Wiley‐Liss, Inc.

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