Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN− Ligands Derive from Tyrosine
Figure 4
Infrared spectra for CpI hydrogenase with isotopically labeled H-cluster containing exogenously bound CO.
The infrared spectra are for the CO-inhibited CpI enzyme harboring an H-cluster produced in the presence of L-tyrosine (CpItyr), L-[2-13C]-tyrosine (CpI2-13C-tyr), L-[1-13C]-tyrosine (CpI1-13C-tyr), and L-[U-13C-15N]-tyrosine (CpIU-13C-15N-tyr). Natural abundance COexo was added to CpItyr and CpI2-13C-tyr, which have intrinsic CO ligands. Conversely, 13COexo was added to CpI1-13C-tyr and CpIU-13C-15N-tyr, which have intrinsic 13CO ligands. Comparing the Hox–COexo spectrum for each CpI sample to its respective Hox spectrum (Fig. 3), shifts of 5–10 cm−1 were observed for the ν(CN) modes and the ν(μ–CO) mode in all four cases. The ν(CO) mode for the Fep–CO ligand did not change. Meanwhile, the ν(CO) mode for the Fed–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 Fed atom. The peak for the ν(CO)symmetric mode is visible at 2015/1970 cm−1 for CO/13CO. The ν(CO)asymmetric mode, however, cannot be distinguished because its vibrational energy is similar to the ν(CO) mode at 1972/1928 cm−1 for the Fep–CO/Fep–13CO adducts. The changes in vibrational energies, indicated by the dashed lines, correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, again confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided. The 13CN/13C15N and 13CO ligands are shown in red and green, respectively, in the molecular diagrams. Vertical scale bars shown at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 3 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox–COexo clusters.