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Faster interprotein electron transfer in a [myoglobin, b?] complex with a redesigned interface.

ABSTRACT: Direct measurements of electron transfer (ET) within a protein-protein complex with a redesigned interface formed by physiological partner proteins myoglobin (Mb) and cytochrome b(5) (b(5)) reveal interprotein ET rates comparable to those observed within the photosynthetic reaction center. Brownian dynamics simulations show that Mb in which three surface acid residues are mutated to lysine binds b(5) in an ensemble of configurations distributed around a reactive most-probable structure. Correspondingly, charge-separation ET from a photoexcited singlet zinc porphyrin incorporated within Mb to the heme of b(5) and the follow-up charge-recombination exhibit distributed kinetics, with median rate constants, k(f)(s) = 2.1 × 10(9) second(-1) and k(b)(s) = 4.3 × 10(10) second(-1), respectively. The latter approaches that for the initial step in photosynthetic charge separation, k = 3.3 × 10(11) second(-1).

SUBMITTER: Xiong P

PROVIDER: S-EPMC4251596 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Faster interprotein electron transfer in a [myoglobin, b⁵] complex with a redesigned interface.

Xiong Peng P Nocek Judith M JM Vura-Weis Josh J Lockard Jenny V JV Wasielewski Michael R MR Hoffman Brian M BM

Science (New York, N.Y.) 20101101 6007

Direct measurements of electron transfer (ET) within a protein-protein complex with a redesigned interface formed by physiological partner proteins myoglobin (Mb) and cytochrome b(5) (b(5)) reveal interprotein ET rates comparable to those observed within the photosynthetic reaction center. Brownian dynamics simulations show that Mb in which three surface acid residues are mutated to lysine binds b(5) in an ensemble of configurations distributed around a reactive most-probable structure. Correspo ...[more]

PMID: 21097931

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Project description: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 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 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) approximately 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 photoinduced singlet ET across the protein-protein interface, (1)k(et) approximately 2 x 10(8) s(-1).

Project description:Formation of a transient [myoglobin (Mb), cytochrome b(5) (cyt b(5))] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b(5)] complex exhibits dynamic docking (DD), with its cyt b(5) partner in rapid exchange at multiple sites on the Mb surface. A triple mutant (Mb(3M)) was designed as part of efforts to shift the electron-transfer process to the simple docking (SD) regime, in which reactive binding occurs at a restricted, reactive region on the Mb surface that dominates the docked ensemble. An electrostatically-guided brownian dynamics (BD) docking protocol was used to generate an initial ensemble of reactive configurations of the complex between unrelaxed partners. This ensemble samples a broad and diverse array of heme-heme distances and orientations. These configurations seeded all-atom constrained molecular dynamics simulations (MD) to generate relaxed complexes for the calculation of electron tunneling matrix elements (T(DA)) through tunneling-pathway analysis. This procedure for generating an ensemble of relaxed complexes combines the ability of BD calculations to sample the large variety of available conformations and interprotein distances, with the ability of MD to generate the atomic level information, especially regarding the structure of water molecules at the protein-protein interface, that defines electron-tunneling pathways. We used the calculated T(DA) values to compute ET rates for the [Mb(wt), cyt b(5)] complex and for the complex with a mutant that has a binding free energy strengthened by three D/E → K charge-reversal mutations, [Mb(3M), cyt b(5)]. The calculated rate constants are in agreement with the measured values, and the mutant complex ensemble has many more geometries with higher T(DA) values than does the wild-type Mb complex. Interestingly, water plays a double role in this electron-transfer system, lowering the tunneling barrier as well as inducing protein interface remodeling that screens the repulsion between the negatively-charged propionates of the two hemes.

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Faster interprotein electron transfer in a [myoglobin, b?] complex with a redesigned interface.

Publications

Faster interprotein electron transfer in a [myoglobin, b⁵] complex with a redesigned interface.

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