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![[Figure 5]](ig5034fig5mag.jpg)
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Figure 5
Determination of residence time of tightly bound water by XF-MS. (a) Rapid mixing combined with 18O-mediated hydroxyl radical labeling to monitor the time-course of exchange of water in cyt c. LC-ESI-MS is used to identify and isolate the modified peptides, targeted MS/MS is used to identify the sites of 18O labeling, and zoom scans are used to quantify the ratio of 18O- versus 16O-labeling at various mixing delays. (b) Zoom scans for singly protonated peptide 61–72 showing the decrease in the abundance of the 2m/z shifted 18O monoisotopic mass (arrow) that corresponds to the water exchange at M65 and Y67 with increase in the mixing delays. (c) Progress curves (circles and error bars) of water exchange for the 18O labeled side-chain residues. The solid line represents the fit to a single exponential function. Residues W59 and F36 have exchange that is complete at the first measurement, while the rates of exchange of C14, C17, F46, Y48, M65, Y67 and M80 are discretely measured. (d) Sites of 18O-modifications are visualized from the crystal structure 1HRC using PyMOL (DeLano Scientific). The 18O-labeled residues (light blue) in and around the heme (light pink) crevices, and the position of residue T78 (gray) and conserved waters (cyan spheres) HOH112, HOH139 are shown in two orientations of the cyt c molecule. Reproduced from Gupta et al. (2012  ). |
![[Figure 5]](ig5034fig5.jpg)
 | JOURNAL OF SYNCHROTRON RADIATION |
ISSN: 1600-5775
