Ultrafast Vibrational Energy Transfer between Protein and Cofactor in a Flavoenzyme.

Samantha J O Hardman,Andreea I Iorgu,Sam Hay,Nigel S Scrutton,Igor V Sazanovich,Derren J Heyes

doi:10.1021/acs.jpcb.0c04929

Abstract

Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond–picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. Here, we use time-resolved infrared (TRIR) spectroscopy in combination with stable “heavy” isotopic labeling (2H, 13C, 15N) of protein and/or cofactor to probe the vibrational energy transfer (VET) between pentaerythritol tetranitrate reductase (a member of the OYE family) and its noncovalently bound flavin mononucleotide (FMN) cofactor. We show that when the FMN cofactor is photoexcited with visible light, vibrational energy is transferred from the flavin to the surrounding protein environment on the picosecond timescale. This finding expands the scope of VET investigation in proteins, which are limited by suitable intrinsic probes, and may have implications in the understanding of the mechanism of recently discovered photoactive flavoenzymes.

Highlights

Protein motions and enzyme catalysis are often linked
The role of “fast” femto- to nanosecond protein motions in enzymatic catalysis is more contentious.[3−5] It is hypothesized that fast bond vibrations support hydride transfer from the nicotinamide (NAD(P)H) coenzyme to the flavin mononucleotide (FMN, Figure 1A) cofactor in the old yellow enzyme (OYE) family of ene-reductases.[6]
It has previously been shown that incorporation of different stable “heavy” isotopes (2H, 13C, 15N) in both the protein scaffold and/or the FMN cofactor of the OYE pentaerythritol tetranitrate reductase (PETNR) influences the kinetics of reductive hydride transfer.[11]

Summary

Introduction

Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond−picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. EADS1 and EADS2 for each isotopologue are virtually identical in shape, but differ in intensity (Figure S7), meaning that the first transition (τ1 ≈ 4.4 ps) does not result in any structural change and can be assigned to FMN excited state relaxation.

Results

Conclusion