Photoinitiated Singlet and Triplet Electron Transfer across a Redesigned [Myoglobin, Cytochrome b5] Interface

We describe a strategy by which reactive binding of a weakly-bound, ‘dynamically 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; 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 fits well with recent proposals that complex formation is a two-step process, proceeding through the formation of a weakly-bound encounter complex (‘diffuse cluster’) to a well-defined bound complex (‘reactive cluster’). 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, koff ≫ ket, to the slow-exchange regime, ket ≫ koff, and both mutants exhibit rapid intracomplex ET from the triplet excited state to Fe3+b5 (rate constant, ket ~ 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 photo-induced singlet ET across the protein-protein interface, 1ket ≈ 2 × 108 s−1.