Orientation selective (OS) RIDME and PELDOR were conducted on a low-spin Co complex coordinated by two nitroxide (NO) labelled 2,2':6',2''-terpyridine ligands. Co-NO RIDME at W- and Q-band gave insight into the relative orientation between the Co-NO interspin vector (r) and the NO moiety. This was further supported by W-band Co-NO PELDOR that also allowed elucidating the relative orientation of the Co and NO g-tensors. Differences to earlier predictions were confirmed by DFT calculations. Finally, NO-NO PELDOR allowed retrieving the mutual orientations between the NO-NO interspin vector (r) and the NO moieties. The results demonstrate that OS-RIDME and -PELDOR can provide geometric structure information on a system containing a Co ion and two nitroxides. Especially, the high sensitivity and ease of interpretation of RIDME at W-band opens avenues for new applications of Co as orthogonal spin label.

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron–electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

The sensitivity of pulsed electron paramagnetic resonance (EPR) measurements on broad-line paramagnetic centers is often limited by the available excitation bandwidth. One way to increase excitation bandwidth is through the use of chirp or composite pulses. However, performance can be limited by cavity or detection bandwidth, which in commercial systems is typically 100-200MHz. Here we demonstrate in a 94GHz spectrometer, with >800MHz system bandwidth, an increase in signal and modulation depth in a 4-pulse DEER experiment through use of composite rather than rectangular π pulses. We show that this leads to an increase in sensitivity by a factor of 3, in line with theoretical predictions, although gains are more limited in nitroxide-nitroxide DEER measurements.

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