This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Carbonic anhydrase (CA) is the most common enzyme in red blood cells. It catalyzes the equilibration between CO2 and carbonic acid, and thus plays a critical role in respiration. Over one hundred crystals structures have been reported for CA and the enzyme has been studied in depth by a variety of biochemical and spectroscopic probes. Despite this extensive investigation, definitive evidence for the protonation state of the solvent molecule bound to the Zn has remained elusive. Recent 67Zn NMR data have suggested that the Zn site does not change with pH, implying that the pH=6.8 transition does not involve protonation of the Zn-bound solvent molecule, but rather must involve protonation at some remote site. We showed last year that high-resolution x-ray emission can be used to probe the structure of the Zn site by using the sensitivity of ligand-to-metal charge-transfer transitions to local structure. We reported preliminary protein consistent with a change in protonation state of the zinc-bound solvent. We have used the latest beam time to repeat these measurements with higher signal/noise, and have confirmed our finding that, at least from the x-ray emission perspective, the Zn-bound solvent molecule in CA does change protonation state.
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