posted on 2023-11-20, 21:20authored byJifu Duan, Astrit Veliju, Oliver Lampret, Lingling Liu, Shanika Yadav, Ulf-Peter Apfel, Fraser A. Armstrong, Anja Hemschemeier, Eckhard Hofmann
[FeFe]-hydrogenases
are efficient H2 converting biocatalysts
that are inhibited by formaldehyde (HCHO). The molecular mechanism
of this inhibition has so far not been experimentally solved. Here,
we obtained high-resolution crystal structures of the HCHO-treated
[FeFe]-hydrogenase CpI from Clostridium pasteurianum, showing HCHO reacts with the secondary amine base of the catalytic
cofactor and the cysteine C299 of the proton transfer pathway which
both are very important for catalytic turnover. Kinetic assays via
protein film electrochemistry show the CpI variant C299D is significantly
less inhibited by HCHO, corroborating the structural results. By combining
our data from protein crystallography, site-directed mutagenesis and
protein film electrochemistry, a reaction mechanism involving the
cofactor’s amine base, the thiol group of C299 and HCHO can
be deduced. In addition to the specific case of [FeFe]-hydrogenases,
our study provides additional insights into the reactions between
HCHO and protein molecules.