Light-Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

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.

Keywords: DEER/PELDOR; EPR spectroscopy; heme proteins; porphyrinoids; triplet state.

Figure 1

(a) Structure of the model peptide based on DFT data.20 The distance between the center of the tetraphenylporphyrin and the N−O midpoint is indicated. (b) Structure of human neuroglobin (PDB: 4MPM).40 The distance between the center of the ZnPP and the average position of the MTSSL rotamers computed with the software MMM (Multiscale Modelling of Macromolecules),44 is indicated. Details are reported in the Supporting Information.

Figure 2

Influence of the zero‐time determination on the distance analysis in LaserIMD and ReLaserIMD measured on the model peptide. For both datasets: pulse sequences (a), raw dipolar time traces with the selected zero time positions used in the distance analysis (b), form factors with the corresponding fits (c) and distance distributions (d) obtained by DeerAnalysis.45 The experimental conditions and the parameters of the data analysis are reported in the Supporting Information.

Figure 3

PDS data measured on ZnG19: (a) LiRIDME and ReLaserIMD pulse schemes, (b) form factors (grey) and best fits to the LiRIDME (azure) and the ReLaserIMD (violet) data and (c) corresponding distances distributions. The distance analyses have been performed with DeerAnalysis, for ReLaserIMD, and with OvertoneAnalysis for LiRIDME (with 50 % contribution of the second harmonic overtone). The error bars have been obtained using the validation procedure, implemented in both softwares, varying the starting point for the background fitting between 300 and 500 ns and adding 50 % of the original noise. The experimental conditions are reported in the Supporting Information.