0428同步年報-2021-全

050 ACTIVITY REPORT 2021 Biradical Mechanism for PQQ-Dependent Hydride Transfer Chemistry High-resolution X-ray crystal-structure analyses with spectral and biochemical studies on methanol dehydrogenase (MDH) from Methylococcus capsulatus (Bath) in various states reveal the biradical mechanism of pyrroloquinoline quinone (PQQ)-dependent hydride transfer chemistry during catalytic turnover. Fig. 1 : Overall structure of apo -MDH complexed with PQQ (left) and a close view at the catalytic binding site (right) with the Cys131-Cys132 center- reduced form. [Reproduced from Ref. 1] 12B2 , together with the properties through electron paramagnetic resonance (EPR), Infrared (IR) and Raman spectral and biochemical studies. They investigated both the holo- and apo- MDH, that is, with and without PQQ, respectively, but with the Cys131-Cys132 center reduced. The high-resolution structures of MDH from M. capsulatus (Bath) were determined in various states of the protein in which the vicinal disulfide bridge near the PQQ cofactor was broken ( Fig. 1 ). IR and Raman spectra performed at TLS 14A1 indicated and confirmed that the broken disulfide bond exists in MDH but was unaffected by the radiation damage of X-rays. The research team presented solid evidence that the disulfide bridge in MDH is redox-active and participates with the PQQ prosthetic group in electron transfers within the protein. In the case of the holo -MDH with the fully reduced disulfide bridge, they observed one-electron reduction of the PQQ by the thiols of Cys131-Cys132 and detected an EPR dipolar spectrum associated with the biradical system formed by the PQQ radical and the disulfide radical anion centered at the disulfide structure. In the X-ray structure of M. capsulatus (Bath) MDH, the two M ethylococcus capsulatus (Bath), a methanotroph, exists in anoxic or anaerobic environments, and metabolizes methane into methanol and subsequently into formaldehyde and other one-carbon (C1) substances. Methanol dehydrogenase (MDH) from M. capsulatus (Bath) is a quinoprotein alcohol dehydrogenase that uses the pyrroloquinoline quinone (PQQ) prosthetic group together with a calcium (Ca 2+ ) ion to catalyze the oxidation of methanol (CH 3 OH), and further to convert it to formaldehyde (HCHO). The active site of MDH contains a rare disulfide bridge between adjacent cysteine residues. This disulfide bridge is clearly seen in the X-ray crystal structure of MDH from M. capsulatus (Bath). As a vicinal disulfide, the structure is highly strained, indicating that it might work together with the PQQ prosthetic group and the Ca 2+ ion in the catalytic turnover during methanol oxidation. Chun-Jung Chen (NSRRC) and his collaborative research teams at Academia Sinica and National Pingtung University analyzed the crystal structures of MDH from M. capsulatus (Bath) under various conditions using X-ray protein crystallography at TPS 05A , TLS 15A1 , SP 44XU and SP