Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases

Abstract

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species ( Br 2D NiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotrons

“…One method exploits the fact that X-rays induce photoelectrons within the crystal bulk solvent to initiate redox reactions at cryogenic temperatures, in a controlled manner using the principle of composite data sets, with the support of UV-Vis absorption microspectrophotometry (Berglund et al, 2002). With more sensitive detectors, full data sets can be used to provide dose points (Rose et al, 2022). Other exciting possibilities include the use of electric field stimulation to target specific protein motions (Hekstra et al, 2016) and that of a nanosecond infrared (IR) laser to induce temperature jumps (Wolff et al, 2023).…”

“…Because the kinetics of a reaction can differ greatly for a protein between the solution and the crystalline state (see Section 4.1), it is of prime importance to perform these spectroscopic experiments directly on the crystals that will be used during the diffraction experiment, and possibly also in solution. A number of instruments and facilities have been built for this purpose, in general close to synchrotron sources, such as the icOS (in crystallo Optical Spectroscopy) Laboratory at the ESRF (von Stetten et al, 2015), the SpectroLab at the SLS (Pompidor et al, 2013) and beamline BL26B1 at SPring-8 (Rose et al, 2022), which took over the online microspectrophotometry capability of BL26B2 (Sakaguchi et al, 2016).…”

Spectroscopically validated multiple structures from one crystal (MSOX) and damage-free atomic structures using XFEL for copper nitrite reductases

“…Structures free from radiation-induced chemistry (FRIC structures) for proteins containing redox centres have become possible using single shot femtosecond pulses from X-ray Free Electron Lasers [3]. We have used high energy X-rays from SACLA to obtain atomic/sub-atomic resolution structures of three different nitrite reductases in a variety of functional states including substrate and product bound species alongside single crystal optical spectra.…”