Pico-meter resolution structure of the coordination sphere in the metal-binding site in a metalloprotein by NMR

Most of our understanding of chemistry derives from atomic-level structures obtained with single crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well defined, with errors in the positions on the order of 10-4-10-5 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10-1 Å. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, since the uncertainty in positions can cover all the known ranges of bond-lengths and bond-angles for a given type of metal-complex. Here we introduce a new approach that enables determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio-frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign 1H, 13C, and 15N resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the CoII coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the non-reactive nature of the CoII/ZnII centers in these proteins, that allows them to play a purely structural role.