The Laser-Induced Potential Jump: A Method for Rapid Electron Injection into Oxidoreductase Enzymes

Abstract

Oxidoreductase enzymes often perform technologically useful chemical transformations using abundant metal cofactors with high efficiency under ambient conditions. The understanding of the catalytic mechanism of these enzymes is, however, highly dependent on the availability of well-characterized and optimized time-resolved analytical techniques. We have developed an approach for rapidly injecting electrons into a catalytic system using a photoactivated nanomaterial in combination with a range of redox mediators to produce a potential jump in solution, which then initiates turnover via electron transfer (ET) to the catalyst. The ET events at the nanomaterial-mediator-catalyst interfaces are, however, highly sensitive to the experimental conditions such as photon flux, relative concentrations of system components, and pH. Here, we present a systematic optimization of these experimental parameters for a specific catalytic system, namely, [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1). The developed strategies can, however, be applied in the study of a wide variety of oxidoreductase enzymes. Our potential jump system consists of CdSe/CdS core–shell nanorods as a photosensitizer and a series of substituted bipyridinium salts as mediators with redox potentials in the range from −550 to −670 mV (vs SHE). With these components, we screened the effect of pH, mediator concentration, protein concentration, photosensitizer concentration, and photon flux on steady-state photoreduction and hydrogen production as well as ET and potential jump efficiency. By manipulating these experimental conditions, we show the potential of simple modifications to improve the tunability of the potential jump for application to study oxidoreductases.

In Situ Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

“…This allows following the evolution of cofactor states by transient IR flash-photolysis spectroscopy with subturnover time resolution . While hydrogenase remain the main target for such experiments, the authors suggest that “the developed strategies can be applied in the study of a wide variety of oxidoreductase enzymes” …”

“…62 While hydrogenase remain the main target for such experiments, the authors suggest that "the developed strategies can be applied in the study of a wide variety of oxidoreductase enzymes". 136…”

Tailoring electron transfer pathway for photocatalytic N2-to-NH3 reduction in a CdS quantum dots-nitrogenase system

“…For this, enzymes are isolated from their biological environment and coupled with QD directly [16][17][18][19][20][21][22][23][24] or using electron transfer mediators. [25][26][27][28] Being the only N 2 reducing enzyme in nature, nitrogenase is of particular interest for the development of a biohybrid because it is capable of the crucial multi-electron N 2 to ammonia reduction with high catalytic rate and selectivity under ambient conditions (Fig. 2).…”

Time-Resolved Infrared Spectroscopy Reveals the pH-Independence of the First Electron Transfer Step in the [FeFe] Hydrogenase Catalytic Cycle

“…Previous studies by our groups established a relationship between pH and the efficiency of the mediator reduction by NRs. 25 As the pH becomes more alkaline, hole transfer from the rod to the sacrificial electron donor becomes faster, resulting in a net increase in ET efficiency to the mediator. These factors culminate in a larger, more negative solution potential jump, which can influence the rate of ET to the enzyme.…”

“…These factors culminate in a larger, more negative solution potential jump, which can influence the rate of ET to the enzyme. 25 To compensate for the pH dependent efficiency, we have carefully attenuated the excitation source to obtain consistent potential jumps at each pH value. Consistent potential jumps were confirmed by TRVis studies monitoring the population of the reduced mediator in solution.…”