Photoinduced Electron Transfer in Eosin-Modified Co(II)-Protoporphyrin IX Reconstituted Myoglobin and α- or β-Hemoglobin Subunits: Photocatalytic Transformations by the Reconstituted Photoenzymes1

A series of hemo-protein-derived photocatalysts, prepared by reconstitution of the respective apo-proteins with Co(II)-protoporphyrin IX and chemical modification of the protein with the eosin chromophore, is presented. Apo-myoglobin, Apo-Mb, was reconstituted with Co(II)-protoporphyrin IX and further modified with eosin-isothiocyanate ( 3 ) to yield the photocatalyst Eo 2 - -Mb-Co(II). The protein is loaded by two eosin chromophore units. Photoexcitation of Eo 2 - -Mb-Co(II) yields the electron transfer species Eo • - -Mb-Co(I) formed by direct oxidative quenching of T Eo 2 - -Mb-Co(II), k q = 5.2 × 10 4 s - 1, and via an indirect path where self-quenching of the eosin-chromophore units yields the intermediate redox products (Eo • 3 - + Eo • - )-Mb-Co(II) that, in the presence of Na 2 EDTA, generate the Eo • - -Mb-Co(I) in a secondary dark electron transfer, k r = 330 s - 1 . The reconstituted protein Eo 2 - -Mb-Co(II) reveals photocatalytic features and its steady-state illumination in the presence of Na 2 EDTA yields hydrogen evolution, φ = 2 × 10 - 4, or photohydrogenation of acetylene to ethylene, φ = 1 × 10 - 2 . The reconstituted photocatalyst Eo 2 - -Mb-Co(II) reveals enzyme-like behavior. Photohydrogenation of acetylenedicarboxylic acid ( 6 ) by Eo 2 - -Mb-Co(II) in the presence of Na 2 EDTA reveals stereospecificity and formation of maleic acid as the hydrogenation product and kinetics that follow the Michaelis−Menten model, K m = 4 mM, V max = 0.6 μM·min - 1 . Similarly, the α- and β-subunits of hemoglobin, Hb, were reconstituted with Co(II)-protoporphyrin IX to yield α-Hb-Co(II) and β-Hb-Co(II). The β-Hb-Co(II) was specifically modified at cysteine 93 residue by eosin maleimide ( 4 ) to form Eo 2 - -β-Hb-Co(II). The α-Hb-Co(II) was modified at a single, unknown, lysine residue by eosin maleimide ( 4 ) to generate Eo 2 - -α-Hb-Co(II). Only oxidative quenching proceeds in Eo 2 - -β-Hb-Co(II) and Eo 2 - -α-Hb-Co(II) to yield the redox photoproducts, Eo • - -β-Hb-Co(I) and Eo • - -α-Hb-Co(I), k q β = 1.8 × 10 3 s - 1 and k q α = 6.5 × 10 3 s - 1, respectively. The back electron-transfer rates of the redox species are k b β = 0.37 × 10 3 s - 1 and k b α = 3.4 × 10 3 s - 1, respectively. The site-specific modification at cysteine 93 residue of β-Hb by the chromophore and the known X-ray structure of β-Hb which defines the electron transfer distance in Eo 2 - -β-Hb, d = 12.87 Å, enabled the analysis of the experimental electron-transfer rate constants according to Marcus theory: λ = 1.1 eV; β = 1.35 Å - 1 . The reorganization energy, λ, associated with the electron transfer in Eo 2 - -α-Hb-Co(II) is similar, λ = 1.15 eV. Application of the β-value extracted for the Eo 2 - -β-Hb-Co(II) system to the Eo 2 - -α-Hb-Co(II) assembly enabled estimation of the electron-transfer distance in the latter system, d = 11.2 Å, and elucidation of the lysine-90 residue as the modification site by the eosin chromophore. The two reconstituted proteins, Eo 2 - -α-Hb-Co(II) and Eo 2 - -β-Hb-Co(II), reveal photocatalytic properties. Their steady-state irradiation in the presence of the Na 2 EDTA resulted in photohydrogenation of acetylene to ethylene, φ α = 0.02; φ β = 0.004. The photogenerated redox species of the reconstituted proteins Eo • - -Mb-Co(I) and Eo • - -α/β-Hb-Co(I) reveal pronounced stabilities against back electron transfer. This was attributed to spatial separation of the redox species by the rigid protein assemblies. The stability of the redox photoproducts Eo • - -Mb-Co(I) enabled tailoring a cyclic photosynthetic assembly where the semisynthetic photoenzyme Eo 2 - -Mb-Co(II) as a reductive biocatalyst is coupled to lactate dehydrogenase, LDH, an oxidative biocatalyst, using N -methylferrocene caproic acid ( 5 ) as diffusional electron mediator. Steady-state irradiation of an assembly composed of Eo 2 - -Mb-Co(II), LDH, and 5 in the presence of acetylene and lactic acid yields the cyclic photoinduced hydrogenation of acetylene by lactic acid to yield ethylene and pyruvic acid, φ = 2 × 10 - 3 . The system mimics artificially the functions of the photosynthetic bacteria chloroflexus.