Interprotein Electron Transfer Between Ferredoxin and Ferredoxin–NADP⁺ Reductase: Electrostatic Steering Versus Conformational Gating in the Encounter Complex

الملخص

The transient complex formed between ferredoxin (Fd) and ferredoxin–NADP⁺ reductase (FNR) constitutes the terminal electron-transfer (ET) step of the photosynthetic electron transport chain, channeling reducing equivalents from Photosystem I to NADP⁺. Whether the rate of interprotein ET is governed predominantly by long-range electrostatic steering during association, or by conformational gating within the encounter complex, remains a central and incompletely resolved question. Here we combine laser flash photolysis, stopped-flow spectroscopy, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), site-directed mutagenesis of charged interface residues, and Brownian and molecular dynamics (MD) simulations to dissect the relative contributions of these two mechanisms in the Anabaena PCC 7119 system at 298 K. We find that the observed first-order ET rate constant k_ET = 5.8 × 10⁴ s⁻¹ at low ionic strength is reduced more than tenfold at high ionic strength (1.0 × 10⁴ s⁻¹ at 500 mM NaCl), confirming a dominant electrostatic contribution to complex formation. Charge-reversal mutants of FNR (E301A, E139K) and Fd (E94K) reduce the bimolecular association rate constant k_on by up to 92%, while leaving the intracomplex ET rate largely unchanged, indicating that electrostatics primarily govern encounter rather than the activated ET step itself. The temperature dependence of k_ET yields an apparent activation enthalpy ΔH‡ = 38.4 kJ/mol that exceeds the Marcus-predicted barrier, and a solvent kinetic isotope effect of 1.6, both consistent with a partially rate-limiting conformational gating step. MD simulations reveal a 1.8 Å reorientation of the FAD isoalloxazine ring and a fluctuating donor–acceptor distance (10.4 ± 0.9 Å) that modulates electronic coupling. We conclude that electrostatic steering governs the rate of encounter-complex formation, whereas conformational gating modulates the productive ET rate within the bound state, and we propose a unified two-step model reconciling decades of apparently conflicting kinetic data.