Binding and Energetics of Electron Transfer between an Artificial Four-Helix Mn-Protein and Reaction Centers from <i>Rhodobacter sphaeroides</i>

The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium <i>Rhodobacter sphaeroides</i> was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to Q_A, the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P⁺, with a dissociation constant of 1.2 μM at pH 9.4, comparable to the binding constant of <i>c</i>-type cytochromes. Amino acid substitutions of surface residues on the Mn-protein resulted in increases in the dissociation constant to 8.3 μM. The extent of reduction of P⁺ by the P1 Mn-protein was dependent on the P/P⁺ midpoint potential and the pH. Analysis of the free energy difference yielded a midpoint potential of approximately 635 mV at pH 9.4 for the Mn cofactor of the P1 Mn-protein, a value similar to those found for other Mn cofactors in proteins. The linear dependence of −56 mV/pH is consistent with one proton being released upon Mn oxidation, allowing the complex to maintain overall charge neutrality. These outcomes demonstrate the feasibility of designing four-helix bundles and other artificial metalloproteins to bind and transfer electrons to bacterial reaction centers and establish the usefulness of this system as a platform for designing sites to bind novel metal cofactors capable of performing complex oxidation–reduction reactions

Mechanism of Triplet Energy Transfer in Photosynthetic Bacterial Reaction Centers

In purple bacterial reaction centers, triplet excitation energy transfer occurs from the primary donor P, a bacteriochlorophyll dimer, to a neighboring carotenoid to prevent photodamage from the generation of reactive oxygen species. The B_B bacteriochlorophyll molecule that lies between P and the carotenoid on the inactive electron transfer branch is involved in triplet energy transfer between P and the carotenoid. To expand the high-resolution spectral and kinetic information available for describing the mechanism, we investigated the triplet excited state formation and energy transfer pathways in the reaction center of <i>Rhodobacter sphaeroides</i> using pump–probe transient absorption spectroscopy over a broad spectral region on the nanosecond to microsecond time scale at both room temperature and at 77 K. Wild-type reaction centers were compared with a reaction center mutant (M182HL) in which B_B is replaced by a bacteriopheophytin (Φ), as well as to reaction centers that lack the carotenoid. In wild-type reaction centers, the triplet energy transfer efficiency from P to the carotenoid was essentially unity at room temperature and at 77 K. However, in the M182HL mutant reaction centers, both the rate and efficiency of triplet energy transfer were decreased at room temperature, and at 77 K, no triplet energy transfer was observed, attributable to a higher triplet state energy of the bacteriopheophytin that replaces bacteriochlorophyll in this mutant. Finally, detailed time-resolved spectral analysis of P, carotenoid, and B_B (Φ in the M182HL mutant) reveals that the triplet state of the carotenoid is coupled fairly strongly to the bridging intermediate B_B in wild-type and Φ in the M182HL mutant, a fact that is probably responsible for the lack of any obvious intermediate ³B_B/³Φ transient formation during triplet energy transfer