Dipole electron-electron resonance spectroscopy (DEER) is a technique that can measure electron separations of 15-80 Å (1) and relative orientations in molecular systems that are challenging for other modalities, such as in large or unstable protein complexes (2).
DEER is typically performed on systems that have had pairs of paramagnetic spin labels covalently attached to cysteine residues of constituent proteins (3, 4). But spin labelling introduces a set of complications. Wild-type proteins may not be amenable to appropriate spin labelling without mutation. The relatively isotropic electron spectrum of most spin labels preclude orientation information from being recovered in a single experiment, and thus multiple spin label pairs can be required to determine it. Mutations and spin labels can affect the structure of protein complexes (2), and flexible spin labels also introduce rotamer distributions into measured distances (5).
Many proteins incorporate paramagnetic centres, however, that can ameliorate these difficulties. No mutation is required to introduce an intrinsic paramagnetic centre, and they generally have well-defined positions. Metal-centred electron spectra are also often highly anisotropic (6), which allows orientation information to be measured without multiple mutants (7).
Unlike most common metal-centred systems, haem-centred proteins are particularly challenging because bandwidth limitations restrict the range of spectral (and therefore molecular) orientations that can be accessed to fully characterise the orientation behaviour of the system, and recent attempts have made no attempt to analyse the system anisotropy (8, 9).
In this work, an unprecedented range of molecular orientations has been sampled and combined with distance information measured with an alternative, non-orientation selective technique (RIDME (10-13)) and orientation-selective simulations to provide both a distance and a relative orientation estimate for the two spin labels in a spin-labelled cytochrome P450.