BEORHN: Biological Enzymatic Oxidation of Reactive Hydroxylamine in Nitrification via Combined Structural Biology and Molecular Simulation

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We aim to gain a full mechanistic understanding of the enzymatic oxidation of hydroxylamine, NH2OH, in methane oxidising and ammonia oxidising bacteria using integrated structural biology and cutting-edge computational techniques. We will study two enzyme families, hydroxylamine oxidoreductase (HAO) and cytochromes P460 (CytP460) that oxidise hydroxylamine to NO and N2O respectively. Although the proteins are unrelated, each have an unusual heme-protein cross-link to a Tyr (HAO) or Lys (CytP460). This study will use closely interleaved experimental and computational methods. Static and time resolved, cryogenic and room temperature crystal structures of different redox states and ligand complexes of P460 proteins and mutants will be determined via X-ray crystallography and single crystal spectroscopy. We will make use of state of the art microfocus synchrotron and XFEL beamlines to measure serial data from microcrystals at room temperature. Spectroscopically-validated structures from the experimental programme will be the starting point for combined quantum mechanical/molecular mechanical (QM/MM) optimizations to fully characterize the redox and protonation states relevant to the native and bound ligands and to explore reaction pathways. The intermediates obtained from crystallography will be used as the starting structures for QM/MM elucidation of the reaction mechanism, using advanced projector-based QM embedding techniques, validating the experimental findings and identifying transient intermediates elusive to experimental determination. Simulated spectra and in silico mutations will be carried out in parallel to identify and understand role of cross-linking, heme deformation and heme environment to reactivity. We will use our mechanistic findings to design and then experimentally characterize mutant enzymes with different product formation, tuning CytP460 to produce NO instead of N2O, and the reverse in HAO.