Fig. 2. FTIR spectra.

Reactions used 100 µM HydG^WT and (¹³C)₉-Tyr, producing ¹³CO and ¹³CN ligands, unless indicated otherwise. (A) SF-FTIR spectra measured at 30 and 1200 s (solid lines) and at 10 s using 800 µM HydG^WT (dotted line, plotted at half intensity). (B) Time dependence of formation and decay of the following species: 4OB^• determined by EPR spectroscopy, two experimental runs (•, °) and corresponding kinetic fit (dashed line) (17); FTIR data (no kinetic fit) of complex A (red) and complex B (blue) determined by the peak heights of their respective ν(CO) modes [see (A)]; and free CO trapped by myoglobin (green) (fig. S3). Each data set is scaled to unity at its maximum value. (C) Comparison of the time dependence of the peak heights of all ν(CO) and ν(CN) bands for complex A and complex B. Complex A red symbols are as follows: °, ν(¹³CN); •, ν(¹³CO). Complex B blue symbols are as follows: °, ν(¹³CN); •, ν(¹³CO) 2010 cm⁻¹; +, ν(¹³CO) 1960 cm⁻¹. The data for complex A were taken from measurements by using 800 µM HydG^WT in order to enhance the signal:noise ratio of the ν(CN) mode. (D) Table of frequencies for observed IR bands of complexes A and B prepared by using Tyr (middle column) or (¹³C)₉-Tyr (right-most column). (E) SF-FTIR spectrum of complex B measured at 900 s and prepared by using a 1:1 mixture of Tyr and ¹³COO-Tyr (top). Average of the ¹²CO and ¹³CO product B spectra (bottom). The arrows indicate new bands not present in either the ¹²CO or ¹³CO spectra of complex B. (F) SF-FTIR spectra of complex B measured at 1200 s and prepared by using (¹³C)₉ ^15N-Tyr (top) and (¹³C)₉Tyr (bottom). Expected (CN) and (CO) bands for ¹³C¹⁵N-containing complex B in the absence of ν(CN)/ ν(CO) vibrational mixing (dotted line). Predicted band shifts computed simply by the change in the reduced mass.