5 research outputs found
Enzymatic Single-Molecule Kinetic Isotope Effects
Ensemble-based measurements of kinetic
isotope effects (KIEs) have
advanced physical understanding of enzyme-catalyzed reactions, but
controversies remain. KIEs are used as reporters of rate-limiting
H-transfer steps, quantum mechanical tunnelling, dynamics and multiple
reactive states. Single molecule (SM) enzymatic KIEs could provide
new information on the physical basis of enzyme catalysis. Here, single
pair fluorescence energy transfer (spFRET) was used to measure SM
enzymatic KIEs on the H-transfer catalyzed by the enzyme pentaerythritol
tetranitrate reductase. We evaluated a range of methods for extracting
the SM KIE from single molecule spFRET time traces. The SM KIE enabled
separation of contributions from nonenzymatic protein and fluorophore
processes and H-transfer reactions. Our work demonstrates SM KIE analysis
as a new method for deconvolving reaction chemistry from intrinsic
dynamics
Direct Evidence of an Excited-State Triplet Species upon Photoactivation of the Chlorophyll Precursor Protochlorophyllide
The
chlorophyll precursor protochlorophyllide (Pchlide), which
is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase,
has unique excited-state properties that facilitate photocatalysis.
Previous time-resolved spectroscopy measurements have implied that
a long-lived triplet state is formed during the excited-state relaxation
of Pchlide, although direct evidence of its existence is still lacking.
Here we use time-resolved electron paramagnetic resonance (EPR) in
combination with time-resolved absorption measurements at a range
of temperatures (10–290 K), solvents, and oxygen concentrations
to provide a detailed characterization of the triplet state of Pchlide.
The triplet decays in a biphasic, oxygen-dependent manner, while the
first reported EPR signature of a Pchlide triplet displays both emissive
and absorptive features and an antisymmetric spectrum similar to other
porphyrin triplet states. This work demonstrates that the Pchlide
triplet is accessible to various cryogenic spectroscopic probes over
a range of time scales and paves the way for understanding its potential
role in catalysis
Enzymatic single-molecule kinetic isotope effects
Ensemble-based measurements of kinetic isotope effects (KIEs) have advanced physical understanding of enzyme-catalyzed reactions, but controversies remain. KIEs are used as reporters of rate-limiting H-transfer steps, quantum mechanical tunnelling, dynamics and multiple reactive states. Single molecule (SM) enzymatic KIEs could provide new information on the physical basis of enzyme catalysis. Here, single pair fluorescence energy transfer (spFRET) was used to measure SM enzymatic KIEs on the H-transfer catalyzed by the enzyme pentaerythritol tetranitrate reductase. We evaluated a range of methods for extracting the SM KIE from single molecule spFRET time traces. The SM KIE enabled separation of contributions from nonenzymatic protein and fluorophore processes and H-transfer reactions. Our work demonstrates SM KIE analysis as a new method for deconvolving reaction chemistry from intrinsic dynamics