Nitric oxide (NO) and nitroxyl (HNO) have important roles in numerous biological processes. Both NO and HNO target and are a part of common biological mechanisms by reacting with heme proteins, among others, via unique and distinct chemistry. Herein, the binding of NO and HNO to the ferrous heme in myoglobin (Mb) and Mb variants is investigated to elucidate the vibrational properties of heme-HNO complexes in detail. Initially, the expression and purification of Sperm Whale myoglobin and variants was accomplished, and these proteins were then utilized to generate both ferrous heme-nitrosyl and -nitroxyl complexes under anaerobic conditions. All complexes were characterized using UV-visible, electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR) spectroscopy, and nuclear resonance vibrational spectroscopy (NRVS) at SPring-8. The ongoing investigation will give further insight into the electronic structure of ferrous heme-nitrosyl and -nitroxyl complexes to shed light on the common and district biological mechanisms of NO and HNO.
Imaging of atomic structure with X-rays is an essential diagnostics tool for modern natural sciences. The best spatial resolution in coherent X-ray imaging of non-periodic nanostructures is limited by overall low cross sections for elastic X-ray scattering. One possible pathway to overcome the present limitations is to extract structural information from inelastic scattering processes which have higher cross sections with X-rays and reach into higher scattering angles. One such process is metal K-shell fluorescence, which can exhibit two-photon interference effects and thus, carries structural maps of the fluorescencing sample. This approach has two significant strengths. First, it relies on X-ray fluorescence which is emitted uniformly into all directions. Second, the structural information is extracted from an element-specific X-ray fluorescence line, which combines high spatial resolution with spectroscopic sensitivity. In theory, incoherent fluorescence imaging can be applied to crystalline and non-crystalline samples and deliver 3D maps of the nanoscale. We confirmed the presence of such speckles and measured a projection of the FEL nano-focus spot on a metal foil.