Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors.

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two nonconserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn and Fe). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analyses that demonstrate the binding of one Fe ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe, suggesting that Fe at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in the Fe-free and Fe-bound states. NMR structural analysis and molecular docking studies suggest that a single Fe ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming an [Fe(SCys)] complex like that observed previously in two-iron superoxide reductases. Binding of Fe to GCAP5 weakens its ability to activate photoreceptor human GC-E by decreasing GC activity >10-fold. Our results indicate a strong Fe-induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.