Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

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.