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New Assay Method Based on Raman Spectroscopy for Enzymes Reacting with Gaseous Substrates
摘要： Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox-dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non-invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2 and also possesses the non-physiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase-mediated reactions simultaneously. Using the proposed system, we confirmed that H2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2/H2O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (kout) was estimated to be 1.9±0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.
关键词： kinetic experiment,H/D exchange reaction,quantitative analysis of gaseous substrates,Raman spectroscopy,non-invasive measurement,[NiFe] hydrogenase
更新于2025-09-23 15:23:52
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The Quantum Efficiency of Charge Transfer Competing Against Non-Exponential Processes: The Case of Electron Transfer from CdS Nanorods to Hydrogenase
摘要： Photoexcited charge transfer from semiconductor nanocrystals to charge acceptors is a key step for photon energy conversion in semiconductor nanocrystal-based light-harvesting systems. Charge transfer competes against relaxation processes within the nanocrystals, and this competition determines the quantum efficiency of charge transfer. The quantum efficiency is a critical design element in photochemistry, but in nanocrystal–acceptor systems its extraction from experimental data is complicated by sample heterogeneity and intrinsically non-exponential excited-state decay pathways. In this manuscript, we systematically explore these complexities using TA spectroscopy over a broad range of timescales to probe electron transfer from CdS nanorods to the redox enzyme hydrogenase. To analyze the experimental data, we build a model that quantifies the quantum efficiency of charge transfer in the face of competing, potentially non-exponential, relaxation processes. Our approach can be applied to calculate the efficiency of charge or energy transfer in any donor–acceptor system that exhibits non-exponential donor decay and any ensemble distribution in the number of acceptors provided that donor relaxation and charge transfer can be described as independent, parallel decay pathways. We apply this analysis to our experimental system and unveil the connections between particle morphology and quantum efficiency. Our model predicts a finite quantum efficiency even when the mean recombination time diverges, as it does in CdS nanostructures with spatially separated electron–hole pairs that recombine with power-law dynamics. We contrast our approach to the widely-used expressions for the quantum efficiency based on average lifetimes, which for our system overestimate the quantum efficiency. The approach developed here is straightforward to implement and should be applicable to a wide range of systems.
关键词： electron transfer,quantum efficiency,semiconductor nanocrystals,non-exponential processes,charge transfer,hydrogenase,CdS nanorods
更新于2025-09-04 15:30:14