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

Abstract

Many enzymes utilize redox-coupled centres for performing catalysis where the centres are used to control and regulatethe transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding thesubstrate i.e. a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyse the reduction of nitrite to nitricoxide (NO), have proved to be a good model system for studying these complex processes including proton-coupledelectron transfer and their orchestration for substrate binding/utilisation [1].X-rays used to collect crystallographic data can in itself result in changes in the redox states of transition metals utilisedby many biological systems including metalloproteins. This disadvantage has been harnessed to drive a complexchemical reaction requiring the delivery of an electron to the active site and recording the structural changesaccompanying catalysis providing a ‘real-time’ structural movie of an enzymatic chemical reaction, a dream ofenzymologist for decades. By coupling MSOX technique with single-crystal and solution optical spectroscopy, weshow that the electron transfer between the T1Cu and T2Cu redox centres is heavily gated and show a structural moviecapturing the bond-breakage, product formation and its release from the catalytic centre for CuNiRs from two organism[2].Structures free from radiation-induced chemistry (FRIC structures) for proteins containing redox centres have becomepossible using single shot femtosecond pulses from X-ray Free Electron Lasers [3]. We have used high energy X-raysfrom SACLA to obtain atomic/sub-atomic resolution structures of three different nitrite reductases in a variety offunctional states including substrate and product bound species alongside single crystal optical spectra. Thesespectroscopically validated very high resolution FRIC structures with unrestrained SHELXL refinement are providingunprecedented level of details including protonation states of key residues in the catalytic pocket. These new resultswill be reported at the Congress.These recent developments would be reviewed with wider applicability in mind.