Photoinitiated singlet and triplet electron transfer across a redesigned [Myoglobin, Cytochrome b ₅] interface

We describe a strategy by which reactive binding of a weakly bound, adynamically docked (DD)'complex without a known structure can be strengthened electrostatically through optimized placement of surface charges, and discuss its use in modulating complex formation between myoglobin (Mb) and cytochrome b₅ (b₅). The strategy employs paired Brownian dynamics (BD) simulations, one which monitors overall binding, the other reactive binding, to examine [X - K] mutations on the surface of the partners, with a focus on single and multiple [D/E - K] charge reversal mutations. This procedure has been applied to the [Mb, b₅] complex, indicating mutations of Mb residues D44, D60, and E85 to be the most promising, with combinations of these showing a nonlinear enhancement of reactive binding. A novel method of displaying BD profiles shows that the 'hits' of b₅ on the surfaces of Mb(WT), Mb(D44K/D60K), and Mb(D44K/D60K/E85K) progressively coalesce into two 'clusters': a 'diffuse' cluster of hits that are distributed over the Mb surface and have negligible electrostatic binding energy and a 'reactive' cluster of hits with considerable stability that are localized near its heme edge, with short Fe-Fe distances favorable to electron transfer (ET). Thus, binding and reactivity progressively become correlated by the mutations. This finding relates to recent proposals that complex formation is a two-step process, proceeding through the formation of a weakly bound encounter complex to a well-defined bound complex. The design procedure has been tested through measurements of photoinitiated ET between the Zn-substituted forms of Mb(WT), Mb(D44K/D60K), and Mb(D44K/D60K/E85K) and Fe³⁺b₅. Both mutants convert the complex from the DD regime exhibited by Mb(WT), in which the transient complex is in fast kinetic exchange with its partners, k _off ≫ k_et, to the slow-exchange regime, k_et ≫ k_off, and both mutants exhibit rapid intracomplex ET from the triplet excited state to Fe³⁺b₅ (rate constant, k _et ≈ 10⁶ s^-1). The affinity constants of the mutant Mbs cannot be derived through conventional analysis procedures because intracomplex singlet ET quenching causes the triplet-ground absorbance difference to progressively decrease during a titration, but this effect has been incorporated into a new procedure for computing binding constants. Most importantly, these measurements reveal the presence of fast photoinduced singlet ET across the protein-protein interface, ¹k_et ≈ 2 - 10⁸ s^-1.