Electrostatic redesign of the [Myoglobin, Cytochrome b ₅] interface to create a well-defined docked complex with rapid Lnterprotein electron transfer

Cyt b ₅ is the electron-carrier "repair" protein that reduces met-Mb and met-Hb to their O ₂-carrying ferroheme forms. Studies of electron transfer (ET) between Mb and cyt b ₅ revealed that they react on a "Dynamic Docking" (DD) energy landscape on which binding and reactivity are uncoupled: binding is weak and involves an ensemble of nearly isoenergetic configurations, only a few of which are reactive; those few contribute negligibly to binding. We set the task of redesigning the surface of Mb so that its reaction with cyt b5 instead would occur on a conventional "simple docking" (SD) energy landscape, on which a complex exhibits a well-defined (set of) reactive binding configuration(s), with binding and reactivity thus no longer being decoupled. We prepared a myoglobin (Mb) triple mutant (D44K/D60K/E85K; Mb(+6)) substituted with Zn-deuteroporphyrin and monitored cytochrome b ₅ (cyt b ₅) binding and electron transfer (ET) quenching of the 3ZnMb(+6) triplet state. In contrast, to Mb(WT), the three charge reversals around the "front-face" heme edge of Mb(+6) have directed cyt b5 to a surface area of Mb adjacent to its heme, created a well-defined, most-stable structure that supports good ET pathways, and apparently coupled binding and ET: both Ka and ket are increased by the same factor of 2 × 102, creating a complex that exhibits a large ET rate constant, k _et = 10 ⁶ 1s ^-1, and is in slow exchange (k _off « k _et). In short, these mutations indeed appear to have created the sought-for conversion from DD to simple docking (SD) energy landscapes.