Light-driven biocatalytic reduction of α,β-unsaturated compounds by ene reductases employing transition metal complexes as photosensitizers

Efficient and cost effective nicotinamide cofactor regeneration is essential for industrial-scale bio-hydrogenations employing flavin-containing biocatalysts such as the Old Yellow Enzymes.


S2.2 Effect of the photosensitizer concentration on OYE activity
Table S2 Impact of changing photosensitizer concentration upon the rates of light-driven biocatalytic reduction of cyclohexen--2--one by PETNR or TOYE.

S1.2 Oligonucleotides and PCR reaction conditions
The following pairs of oligonucleotide sequences were used to generate PETNR variants. Nucleotides highlighted in red are mutations of the original sequence, and the triplet codon in bold indicates the subsequent amino acid modified.  [a] Determined after 120 min. [b] Determined by GC analysis after 240 min, except reactions indicated by * which were analysed after 60 min.

S2.2 Effect of the photosensitizer concentration on OYE activity
Given the successful photosensitizer--driven bioreduction of cyclohexen--2--one by both TOYE and PETNR, reaction optimisation studies were performed to enhance TOF and ultimately product yields. Using [Ru(bpz) 2 (dClbpy)]Cl 2 , reactions were performed to determine the optimal levels of photosensitizer in the presence of an excess of TEA and [MV 2+ ]Cl 2 (Table S2 and Figure S4). For both OYEs, product yield increased with photosensitizer concentration, with an optimal concentration of 20 µM ( Figure S5a). [a] Determined after 120 min. [b] Determined by GC analysis after 240 min. Interestingly, while maximal cyclohexanone production was achieved with only 20 mM photosensitizer, the TOF continues to increase at higher levels. This suggests the rate of MV +• formation is sufficient with 20 µM photosensitizer to achieve maximal product generation within 120 min, but the overall catalytic turnover is limited by non--saturating MV +• formation. At higher photosensitizer concentrations, the rate of MV +• formation may also show a greater dependence on other factors, such as the concentration of MV 2+ /TEA and the binding of MV +• .

S2.3 Effect of the sacrificial electron donor concentration on OYE activity
The yields of cyclohexanone produced by OYEs using either [Ru(bpz) 2 (dClbpy)] 2+ or [Ir(Me--2,2ʹ′-bpy) 2 (bpy)] 3+ depend on the concentration of the sacrificial electron donor TEA (Table S3 and Figure  S6). However, in each case, maximal product yields are obtained with 25 mM TEA ( Figure  S5b). These results are consistent with the proposed mechanism where turnover is limited by the rate of generation of the reduced sensitizer upon quenching of the excited state by TEA (Scheme 2a). This is in contrast to direct light--driven flavin reduction mechanisms of PAMO--P3 and YqjM, which both exhibit initial rates independent of the donor concentration. 2,3  [a] Determined after 120 min.  [a] Determined after 120 min. [b] Determined by GC analysis after 240 min, except reactions indicated by * which were analysed after 60 min.

S2.4 pH dependence of OYE activity
The pH dependence of the reaction was determined in TEA buffer solutions (pH 6-10) using two photosensitizers ([Ru(bpz) 2 (dClbpy)]Cl 2 or Ir(Me--2,2ʹ′--bpy) 2 (bpy)]Cl 3 ) and TOYE or PETNR R324C (Table  S4 and Figure S7). In all instances, maximal product yield is obtained in the pH range 8-10 ( Figure  S5c). However the TOFs suggest the optimum pH values lie between 9 and 10 (Table S4). These results are consistent with prior studies showing that NAD(P)H--mediated reactions of PETNR have a broad pH activity profile, with lower conversion rates observed at pH < 7. 4 In the present system, if enzyme deactivation were the sole contributor to the poor performance at low pH, an accumulation of MV +• would be expected due to perturbation of oxidative quenching by FMN. However, no significant concentration of MV +• was detected during reactions at pH 6-7. Therefore, the pH dependency may in part be attributed to protonation of TEA. The TEA cation, formed on reductive quenching of the excited complex, is also subject to an acid--base equilibrium in solution. At low pH, the cationic form persists in solution and may act as an oxidant towards MV +• , thus further perturbing the forward electron transfer within the catalytic cycle. 5 Similar observations and rationale have been applied to systems employing EDTA as a sacrificial donor. 6 The [Ru(bpz) 2 (L-L)] 2+ complexes show a greater pH dependence than the Ir(III) compounds, with a dramatic decrease in activity with increasing acidity of the buffer. This can be ascribed to the deactivation of the photosensitizer upon protonation of the uncoordinated N atoms of the bpz ligands. The ligand--centred radical that is formed upon reductive quenching of the excited sensitizer may be readily protonated at sufficiently low pH to form the conjugate acid [Ru(bpz) 2 (•bpzH)] 2+ , which has pK a ≈7.1. 8 As a consequence, the redox potentials of the complex undergo an anodic shift of ca. 0.2 V, becoming insufficient to reduce MV 2+ . 8 This explains the near inactivity of the system at pH 6, as at this point the majority of the complex is expected to be protonated.