Abstract
To understand design principles for assembling photosynthetic biohybrids that incorporate precisely-controlled sites for electron injection into redox enzyme cofactor arrays, we investigated the influence of chirality in assembly of the photosensitizer ruthenium(II)bis(2,2′-bipyridine)(4-bromomethyl-4′-methyl-2,2′-bipyridine), Ru(bpy)2(Br-bpy), when covalently conjugated to cysteine residues introduced by site-directed mutagenesis in the triheme periplasmic cytochrome A (PpcA) as a model biohybrid system. For two investigated conjugates that show ultrafast electron transfer, A23C-Ru and K29C-Ru, analysis by circular dichroism spectroscopy, CD, demonstrated site-specific chiral discrimination as a factor emerging from the close association between [Ru(bpy)3]2+ and heme cofactors. CD analysis showed the A23C-Ru and K29C-Ru conjugates to have distinct, but opposite, stereoselectivity for the Λ and Δ-Ru(bpy)2(Br-bpy) enantiomers, with enantiomeric excesses of 33.1% and 65.6%, respectively. In contrast, Ru(bpy)2(Br-bpy) conjugation to a protein site with high flexibility, represented by the E39C-Ru construct, exhibited a nearly negligible chiral selectivity, measured by an enantiomeric excess of 4.2% for the Λ enantiomer. Molecular dynamics simulations showed that site-specific stereoselectivity reflects steric constraints at the conjugating sites and that a high degree of chiral selectivity correlates to reduced structural disorder for [Ru(bpy)3]2+ in the linked assembly. This work identifies chiral discrimination as means to achieve site-specific, precise geometric positioning of introduced photosensitizers relative to the heme cofactors in manner that mimics the tuning of cofactors in photosynthesis.
Highlights
Photosynthetic biohybrids created by the integration of synthetic photosensitizers within multi-cofactor redox proteins and enzymes are of growing interest because of the opportunities to use light-initiated single electron transfer chemistry to track intramolecular electron transfer pathways along cofactor arrays, and to follow intermediates during the charge-accumulating redox steps in hydrogen and nitrogen fixation reaction cycles (Brown et al 2016; King 2018; Lam et al 2016; Lee et al 2018; Mulfort and Utschig 2016; Utschig et al 2015)
To gain insight into the linked structures underpinning the position-dependent Photo-induced electron transfer (PET) rates, we investigated structural aspects of Ru(bpy)2(Br-bpy) conjugation by circular dichroism spectroscopy (CD) and molecular dynamics simulations (MD)
The major peaks for the photosensitizer are seen in the conjugate, the ligand-centered π–π* transition (LCT) is located in the near-UV region at about 286 nm, and the metal-to-ligand transition (MLCT) in visible region is discernible as the shoulder on the high energy side of the heme’s Soret band around 457 nm
Summary
Photosynthetic biohybrids created by the integration of synthetic photosensitizers within multi-cofactor redox proteins and enzymes are of growing interest because of the opportunities to use light-initiated single electron transfer chemistry to track intramolecular electron transfer pathways along cofactor arrays, and to follow intermediates during the charge-accumulating redox steps in hydrogen and nitrogen fixation reaction cycles (Brown et al 2016; King 2018; Lam et al 2016; Lee et al 2018; Mulfort and Utschig 2016; Utschig et al 2015). Site-dependent stereoselectivity has been observed in the labeling redox proteins with ruthenium (II) polypyridine complexes and was applied to both heme proteins (Dmochowski et al 2000; Luo et al 1998) and proteolytic enzymes (Haquette et al 2010) These studies demonstrate that complementary geometric shapes and non-covalent interactions within a protein binding pocket can be used to create a conformationally determined, lock-and-key specificity for photosensitizer integration within protein host matrices. Investigations of photosynthetic biohybrid designs for light-harvesting have demonstrated that chiral linkages can be used to create sterically-constrained, chromophore-protein couplings that function to enhance light-harvesting by modulating nuclear relaxation dynamics, extending excited-state lifetimes, and controlling Stokes shift energy losses (Delor et al 2018). These designed protein-chromophore interactions are anticipated to mimic those functioning in native photosynthetic light-harvesting proteins
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