Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN)2] in Polar and Protic Solvents

The [CpFe(CO)(CN)2]− unit is an excellent structural model for the Fe(CO)(CN)2 moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump–probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)2] (M1) in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of M1 in H2O, D2O, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN)2 unit. Comparisons of the spectroscopic and dynamic data obtained for M1 in solution and in a dry film with those obtained for the enzyme led to the conclusion that the protein backbone forms an important part of the bimetallic active site environment via secondary coordination sphere interactions.

Table S3.Spectral diffusion constants (ps) for vCO of M1 obtained in the different solvent by the nodal line slope method.S11 coupling between the asymmetric and symmetric vCO modes.The compound showed the presence of M1 as impurity which is highlighted in the 2D-IR spectrum by the circle.

Table S2. IR frequencies for for the vCO and vCN modes of M1 in mixtures of DMSO/H2O obtained from
Gaussian fitting of the experimental FTIR spectra.All spectroscopic values are given in cm -1 .CN1 refers to the antisymmetric stretch and CN2 refers to the symmetric one.Table S3.Spectral diffusion constants (ps) obtained in the different solvent by the nodal line slope method by fitting a monoexponential funtion to the data as shown in Figure S13.

Figure S13 .
Figure S13.Temporal dependence of the nodal line slope between the 0-1 and 1-2 transition for the vCO to obtain a qualitative measure of the frequency fluctuation correlation function in the different solvents.S13

Figure S14 .S3Figure S1 .
Figure S14.Anisotropy decays for the CO and CN modes in the different solvents.S13

Figure
Figure S2.1 H-13 C HMQC of M1 in D2O displaying cross peaks between the resonance for the C5H5 (Cp) ligand in the 1 H spectrum and the corresponding carbon peak in the 13 C spectrum as well as cross peaks to the CO and the CN ligands.

Figure S7 .Figure S8 .Figure S9 .Figure S10 .
Figure S7.Slices through the 2D-IR spectrum in H2O (top) and MeOH (bottom) under parallel (a) and perpendicular (b) polarization conditions.The pump frequency in each case is given in the legend.Numbers correspond to those used in the main text.

Figure
Figure S11.a) FT-IR b) IR Pump-IR probe c) 2D-IR spectra of CpFe(CO)2(CN) in CH3CN.The blue dotted rectangles highlight couplings between the vCO modes and the vCN modes, while the red one shows the

Figure S13 .
Figure S13.Temporal dependence of the nodal line parameter to obtain the time-dependence measure of the frequency fluctuation correlation function for vCO mode of M1.The red line is a monoexponential fit to the experimental data.a )H2O; b) D2O, c) MeOH, d )DMSO.

Figure S14 .
Figure S14.Anisotropy decays for the CO and CN modes in the different solvents.