Light-Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein.

Maria Giulia Dal Farra,Caterina Martin,Sabine Richert,Charles Larminie,Marilena Di Valentin,Elisabetta Bergantino,Christiane R Timmel,Alice M Bowen,Marina Gobbo

doi:10.1002/cphc.201900139

Abstract

Light‐induced pulsed EPR dipolar spectroscopic methods allow the determination of nanometer distances between paramagnetic sites. Here we employ orthogonal spin labels, a chromophore triplet state and a stable radical, to carry out distance measurements in singly nitroxide‐labeled human neuroglobin. We demonstrate that Zn‐substitution of neuroglobin, to populate the Zn(II) protoporphyrin IX triplet state, makes it possible to perform light‐induced pulsed dipolar experiments on hemeproteins, extending the use of light‐induced dipolar spectroscopy to this large class of metalloproteins. The versatility of the method is ensured by the employment of different techniques: relaxation‐induced dipolar modulation enhancement (RIDME) is applied for the first time to the photoexcited triplet state. In addition, an alternative pulse scheme for laser‐induced magnetic dipole (LaserIMD) spectroscopy, based on the refocused‐echo detection sequence, is proposed for accurate zero‐time determination and reliable distance analysis.

Highlights

Light-induced pulsed Electron paramagnetic resonance (EPR) dipolar spectroscopic methods allow the determination of nanometer distances between paramagnetic sites
EPR techniques that allow the measurement, via the dipolar electron-electron coupling between two paramagnetic species, of distances and distance distributions
Structural information in the range between 1.6 and 8 nm is obtained with high precision and reliability, while the limit of 16 nm is reached under full deuteration of the sample and solvent.[4,5,6]

Summary

University of Groningen

While LiDEER uses the triplet signal for detection and depends on the transverse relaxation time of the triplet, (Re)LaserIMD, using the stable radical for observation, is influenced by the phase memory time of this species This is the reason why, in the specific case of neuroglobin, where the phase memory time of ZnPP triplet state is of the order of 500 ns only, the use of the LiDEER is almost precluded, despite the favorable spin polarization of the triplet (see Figure S2 in the Supporting Information).[47]. In this work we demonstrate that an accurate determination of distance distributions can be achieved using the triplet state of ZnPP coupled to a nitroxide spin label in human neuroglobin This is the first time that the feasibility of the dipolar experiment has been demonstrated for a paradigmatic protein belonging to the class of the hemeproteins, making clear use of the photoexcited triplet state.

Experimental Section

Findings

Conflict of Interest