Photoinitiated Singlet and Triplet Electron Transfer across a Redesigned [Myoglobin, Cytochromeb₅] Interface

Abstract

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 b 5 (b 5 ). 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, b 5 ] 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 b 5 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 Fe 3+ b 5 . 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 Fe 3+ b 5 (rate constant, k et ~ 10 6 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, 1 k et ≈ 2 × 10 8 s −1 .

The key role of water in the dioxygenase function of Escherichia coli flavohemoglobin

“…In most cases reduction is performed by another protein (the electron donor) that reduces the heme through interprotein ET. For the extensively studied mammalian Mb and Hb the reduction partner is Cytochrome-b5 (Cb5) another small heme protein [35][36][37]65]. An interesting question then arises as if the electron entry point to the globin domain is similar in interprotein (Mb/Cb5) and intraprotein (GD/FBD) ET.…”

“…Our results show that in FHb the electron enters the GD through the heme propionates. Interestingly, the results for the Mb/Cb5 show that best ET paths are those were electrons enter directly to Mb though the heme exposed edge which includes the propionates [35][36][37]65]. Although so far only these two cases have been analyzed and there is a complete lack of knowledge of many globin (particularly bacterial globins) reduction partners, it is tempting to propose that redox partners should dock close to the heme exposed edges for efficient ET to occur.…”

“…Then one electron from the reduced Flavin will reduce the heme, while remaining in the semi reduced radical state, until the heme is oxidized after an NOD cycle. FHbs are thus unique globins, since reduction involves an intra-protein ET between domains, and not inter-protein ET, as is the case for Mb and Hb reduction by Cytochrome-b5 [35][36][37]. Although understanding how structure, dynamics and domain-domain interactions in FHbs modulate ET could give insights into general reduction mechanisms of globins, the molecular details of the ET process in FHbs are still unknown.…”

Faster Interprotein Electron Transfer in a [Myoglobin, b 5 ] Complex with a Redesigned Interface

“…We recently described a strategy for redesigning the interface between weakly bound (dynamically docked) ET partner proteins, thereby enhancing and coupling both their binding and reactivity, by optimizing the placement of surface charges within the putative docking interface (6). We applied this strategy to the [Mb, b 5 ] complex and obtained a triple mutant, in which three acidic surface residues surrounding the Mb (horse) heme (Asp 44 , Asp 60 , and Glu 85 ) are replaced by Lys [Mb(+6)], that exhibits both strongly enhanced binding to b 5 (bovine) and intracomplex ET from the triplet state of Zn-deuteroporphyrin (ZnD)–substituted Mb to the Fe 3+ b 5 heme (Fe 3+ P).…”

“…The 3 ZnD that remains after the 1 ZnD photo-cyle is complete undergoes 3 ZnD → Fe 3+ P ET within the complex (6). This charge separation is three orders of magnitude slower than for 1 ZnD, but still slightly faster than the corresponding process in other Zn-substituted protein ET pairs (Table 1).…”