Infrared spectra for CpI hydrogenase with isotopically labeled H-cluster containing exogenously bound CO. Jon M. Kuchenreuther Simon J. George Celestine S. Grady-Smith Stephen P. Cramer James R. Swartz 10.1371/journal.pone.0020346.g004 https://plos.figshare.com/articles/figure/_Infrared_spectra_for_CpI_hydrogenase_with_isotopically_labeled_H_cluster_containing_exogenously_bound_CO_/440583 <p>The infrared spectra are for the CO-inhibited CpI enzyme harboring an H-cluster produced in the presence of L-tyrosine (CpI<sup>tyr</sup>), L-[2-<sup>13</sup>C]-tyrosine (CpI<sup>2-13C-tyr</sup>), L-[1-<sup>13</sup>C]-tyrosine (CpI<sup>1-13C-tyr</sup>), and L-[U-<sup>13</sup>C-<sup>15</sup>N]-tyrosine (CpI<sup>U-13C-15N-tyr</sup>). Natural abundance CO<sub>exo</sub> was added to CpI<sup>tyr</sup> and CpI<sup>2-13C-tyr</sup>, which have intrinsic CO ligands. Conversely, <sup>13</sup>CO<sub>exo</sub> was added to CpI<sup>1-13C-tyr</sup> and CpI<sup>U-13C-15N-tyr</sup>, which have intrinsic <sup>13</sup>CO ligands. Comparing the H<sub>ox</sub>–CO<sub>exo</sub> spectrum for each CpI sample to its respective H<sub>ox</sub> spectrum (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020346#pone-0020346-g003" target="_blank">Fig. 3</a>), shifts of 5–10 cm<sup>−1</sup> were observed for the <i>ν</i>(CN) modes and the <i>ν</i>(μ–CO) mode in all four cases. The <i>ν</i>(CO) mode for the Fe<sub>p</sub>–CO ligand did not change. Meanwhile, the <i>ν</i>(CO) mode for the Fe<sub>d</sub>–CO moiety was replaced with two peaks resulting from symmetric and asymmetric coupled vibrational stretches, as two CO molecules of equal mass are coordinated to the Fe<sub>d</sub> atom. The peak for the <i>ν</i>(CO)<sub>symmetric</sub> mode is visible at 2015/1970 cm<sup>−1</sup> for CO/<sup>13</sup>CO. The <i>ν</i>(CO)<sub>asymmetric</sub> mode, however, cannot be distinguished because its vibrational energy is similar to the <i>ν</i>(CO) mode at 1972/1928 cm<sup>−1</sup> for the Fe<sub>p</sub>–CO/Fe<sub>p</sub>–<sup>13</sup>CO adducts. The changes in vibrational energies, indicated by the dashed lines, correlate with expected changes for <i>ν</i>(<sup>13</sup>CO), <i>ν</i>(<sup>13</sup>CN), and <i>ν</i>(<sup>13</sup>C<sup>15</sup>N) modes, again confirming that the CO and CN<sup>−</sup> ligands are synthesized from tyrosine. Labels indicating the assigned <i>ν</i>(CO) and <i>ν</i>(CN) vibrational modes are provided. The <sup>13</sup>CN/<sup>13</sup>C<sup>15</sup>N and <sup>13</sup>CO ligands are shown in red and green, respectively, in the molecular diagrams. Vertical scale bars shown at 1740 cm<sup>−1</sup> represent a difference of 0.5 milliabsorbance units. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020346#pone-0020346-t003" target="_blank">Table 3</a> summarizes the vibrational energies and corresponding assigned <i>ν</i>(CN) and <i>ν</i>(CO) modes for the H<sub>ox</sub>–CO<sub>exo</sub> clusters.</p> 2011-05-31 00:09:43 spectra cpi hydrogenase isotopically labeled h-cluster containing exogenously bound