posted on 2023-11-08, 20:00authored byRomie
C. Nguyen, Ian Davis, Medhanjali Dasgupta, Yifan Wang, Philipp S. Simon, Agata Butryn, Hiroki Makita, Isabel Bogacz, Kednerlin Dornevil, Pierre Aller, Asmit Bhowmick, Ruchira Chatterjee, In-Sik Kim, Tiankun Zhou, Derek Mendez, Daniel W. Paley, Franklin Fuller, Roberto Alonso Mori, Alexander Batyuk, Nicholas K. Sauter, Aaron S. Brewster, Allen M. Orville, Vittal K. Yachandra, Junko Yano, Jan F. Kern, Aimin Liu
The P450 enzyme CYP121 from Mycobacterium
tuberculosis catalyzes a carbon–carbon
(C–C) bond coupling cyclization of the dityrosine substrate
containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in
less than 5 ms in the rapid freeze-quenching study of CYP121 during
the shunt reaction with peracetic acid or hydrogen peroxide in acetic
acid solution. We show that this intermediate can also be observed
in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing
method for time-resolved serial femtosecond crystallography with X-ray
free-electron laser (tr-SFX-XFEL) technology covering the millisecond
time domain and without freezing, we structurally monitored the reaction
in situ at room temperature. After a 200 ms peracetic acid reaction
with the cocrystallized enzyme–substrate microcrystal slurry,
a ferric-hydroperoxo intermediate is observed, and its structure is
determined at 1.85 Å resolution. The structure shows a hydroperoxyl
ligand between the heme and the native substrate, cYY. The oxygen
atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion.
The end-on binding ligand adopts a near-side-on geometry and is weakly
associated with the iron ion, causing the unusual high-spin state.
This compound 0 intermediate, spectroscopically and structurally observed
during the catalytic shunt pathway, reveals a unique binding mode
that deviates from the end-on compound 0 intermediates in other heme
enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound
cYY, suggesting an active oxidant role of the intermediate for direct
substrate oxidation in the nonhydroxylation C–C bond coupling
chemistry.