Chae Un Kima,b,1, HyoJin Songc, Balendu Sankara Avvarud, Sol M. Grunera,e, SangYoun Parkc,1, and Robert McKennad,1
aCornell High Energy Synchrotron Source, Cornell University, Ithaca, NY 14853;
bDepartment of Physics, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea;
cSchool of Systems Biomedical Science, Soongsil University, Seoul 06978, Republic of Korea;
dDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610;
eDepartment of Physics, Cornell University, Ithaca, NY 14853
Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3-. Previously, the X-ray crystal structures of CO2-bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo- [T = 0 s (CO2-bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the “slow” release of CO2. Two active site waters, WDW (deep water) and WDW′ (this study), replace the vacated space created on CO2 release, and another water, WI (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away (“out”) to pointing toward (“in”) active site rotameric conformation. This study provides for the first time, to our knowledge, structural “snapshots” of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO2 is released. Comparison of the holo- and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion.
carbonic anhydrase II, CO2 release, intermediate states, high-pressure cryocooling
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Author contributions: C.U.K. designed research; C.U.K. and B.S.A. performed research; C.U.K. and S.P. analyzed data; and C.U.K., H.S., B.S.A., S.M.G., S.P., and R.M. wrote the paper.