Into The Blue: A Novel Three-Domain Copper-Nitrite Reductase with a SerCAT residue at the active site

OPPERMAN, DIEDERIK JOHANNES; BRONDINO, CARLOS D.; FERRONI FELIX MARTÍN

Cape Town

Workshop; CCP4 Crystallographic School in South Africa. Data Collection to Structure Refinement and Beyond. Digital Conference; 2021

University of Cape Town

Homotrimeric two-domain NirKs receive electrons from cupredoxins (azurins/pseudoazurins) or cytochrome c550, depending on the microorganism.These groups of enzymes harbours two copper centres at the two-domain core structure: i) an electron transfer centre (T1Cu) that receives the electrons from the physiological electron donor, and ii) a catalytic centre (T2Cu) where the reduction of nitrite to NO takes place. The process can be defined as proton-coupled electron transfer (PCET). Nitite binds to the T2Cu and triggers the release of one electron from T1Cu. T1Cu and T2Cu are connected by the Cys-His bridge and the so called ?sensing loop?.There are two kind of proton channels: the primary proton channel (PPC) that involves a HisPPC on the surface, and ii) a secondary proton channel (SPC) that involves several residues within the substrate channel (SC). All of them finish involving a molecule of water (Wap) (T2Cu active site). Two key residues are relevant for the catalytic cycle: AspCAT and HisCAT. These residues are connected by a bridging water (WB). Other residues are relevant for nitrite accommodation (Ile/ValCAT) and catalytic support at T2Cu active site (Glu and Thr; ET). A complete mechanism was elucidated by combining synchrotron radiation crystallography (SRX) and serial femtosecond crystallography (SFX) with both resting state and nitrite complex enzyme.Recently, three-domain NirKs were discovered and characterized. An extra domain (cupredoxin or cytochrome c) is fused to the two-domain core structure. We recently discovered a new three-domain NirK with a SerCAT residue at the active site opening new interrogants in the field.