MicroED structure of a protoglobin reactive carbene intermediate

Microcrystal electron diffraction (MicroED) is an emerging technique which has shown great potential for describing new chemical and biological molecular structures. [1] Several important structures of small molecules, natural products and peptides have been determined usingab initiomethods. [2] However, only a couple of novel protein structures have thus far been derived by MicroED. [3, 4] Taking advantage of recent technological advances including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of anAeropyrum pernixprotoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an alphahelix ofApePgb into a dynamic loop making the catalytic active site more readily accessible. After exposing the tiny crystals to substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineeredApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enables investigation of structures that were previously beyond reach.