Metalloproteins {perform|carry out|execute} a diverse array of redox-related reactions facilitated by the {increased|elevated|improved|enhanced} chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes {that are|which are|which can be|which might be|that happen to be} {responsible|accountable} for the breakdown of recalcitrant polysaccharides {via|by way of|through|by means of} oxidative cleavage {at the|in the} glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains {to be|to become} established. Neutron protein crystallography which permits direct visualization of protonation states was {used|utilized|employed|utilised|applied|made use of} to investigate the initial {steps|actions|measures|methods} of oxygen activation {directly|straight} following active {site|website|web site|internet site|web-site|web page} copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate {in a|inside a|within a} mixture of superoxo and hydroperoxo states, and we {identify|determine|recognize} the conserved second coordination shell residue His157 {as the|because the} proton donor. Density functional theory (DFT) calculations indicate that {both|each} active {site|website|web site|internet site|web-site|web page} states are {stable|steady}. The hydroperoxo formed is potentially an intermediate {in the|within the|inside the} mechanism of hydrogen peroxide formation {in the|within the|inside the} absence of substrate. We establish that the N-terminal amino group {of the|from the|in the|on the|with the|of your} copper coordinating His1 remains doubly protonated {directly|straight} following molecular oxygen reduction by copper. Aided by mining minima {free|totally free|free of charge|cost-free|absolutely free|no cost} {energy|power} calculations we establish His157 conformational flexibility in {solution|answer|remedy|resolution|option} {that is|that’s|which is|that is certainly|that is definitely|that may be} abolished by steric hindrance {in the|within the|inside the} crystal. A neutron crystal structure of NcLPMO9D at low pH supports occlusion {of the|from the|in the|on the|with the|of your} active {site|website|web site|internet site|web-site|web page} which prevents protonation of His157 at acidic {conditions|circumstances|situations}. 62972-61-6 Data Sheet (R)-2-amino-1-phenylethan-1-ol custom synthesis PMID:24211511
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