Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography

Abstract

Copper-containing nitrite reductases (CuNiRs) that convert NO₂⁻ to NO via a Cu_CAT–His–Cys–Cu_ET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that Asp_CAT and His_CAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D₂O) is positioned with one deuteron directed towards Asp_CAT O^δ1 and one towards His_CAT N^∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D₂O molecule as opposed to D₃O⁺ or OD⁻, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated Asp_CAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with Asp_CAT. Upon the binding of NO₂⁻ a proton is transferred from the bridging water to the O^δ2 atom of Asp_CAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from Asp_CAT to the bound nitrite, enabling the reaction to proceed.