A Combined Far‐Infrared Spectroscopic and Electrochemical Approach for the Study of Iron–Sulfur Proteins

Abstract

Herein, we present the development of a far-infrared spectroscopic approach for studying metalloenzyme active sites in a redox-dependent manner. An electrochemical cell with 5 mm path and based on silicon windows was found to be appropriate for the measurement of aqueous solutions down to 200 cm(-1) . The cell was probed with the infrared redox signature of the metal-ligand vibrations of different iron-sulfur proteins. Each Fe-S cluster type was found to show a specific spectral signature. As a common feature, a downshift of the frequency of the Fe-S vibrations was seen upon reduction, in line with the increase of the Fe-S bond. This downshift was found to be fully reversible. Electrochemically induced FTIR difference spectroscopy in the far infrared is now possible, opening new perspectives on the understanding of metalloproteins in function of the redox state.

The Conformational Changes Induced by Ubiquinone Binding in the Na+-pumping NADH:Ubiquinone Oxidoreductase (Na+-NQR) Are Kinetically Controlled by Conserved Glycines 140 and 141 of the NqrB Subunit

“…The spectral region 1800 -1200 cm Ϫ1 is shown because it contains information on conformational changes in the backbone of the protein, the flavin cofactors, ubiquinone, and amino acid side chains while omitting contributions from the phosphate buffer that occur below 1200 cm Ϫ1 . Fe-S contributions from the iron-sulfur cluster are expected at even lower spectral frequencies (37). Key spectral features that confirm the presence of redox-active flavins include the large positive peak at 1541 cm Ϫ1 due to a redox-induced change in the (CAC) stretch, the large negative peak at 1405 cm Ϫ1 , which shows coupled ␦(C-H) in-plane /␦(N-H) in-plane bending, and the fingerprint of positive and negative peaks in the amide I region between 1600 and 1700 cm Ϫ1 (peaks and attributions are summarized in Table 2) (38).…”

Distinguishing Protonation States of Histidine Ligands to the Oxidized Rieske Iron–Sulfur Cluster through 15N Vibrational Frequency Shifts

“…Calculated LS vibrational frequencies for modes concentrated on the iron-sulfurc ore have an root-mean-square deviation (rmsd) of 10.6 cm À1 from the availablee xperimentally obtained frequencies of Rotsaert et al [42] and an rmsd of 4.8 cm À1 from the available experimentally obtained frequencies of El Khoury et al [43] This result is improved over the BS vibrational frequencies, which have corresponding rmsd values of 16.2 and 5.5 cm .A dditionally,t his is slightly smaller than the rmsd values of 12.5 and 12.2 cm À1 for published BS results obtained by using the BLYP/DZPm ethod. [44] Vibrational frequencies were also calculated for 15 Ni sotopic substitutions within the imidazole rings of the structures described in Figure 2.…”

Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins

“…The concept of the transmission cell is not significantly changed for the mid-infrared (MIR) spectral range. In the far infrared (below 600 cm -1 ), where metal-ligand vibrations can be observed, a cell with silicon or diamond windows was created on the basis of the same cell geometry (El Khoury and Hellwig , 2011 ;Marboutin et al , 2011 ). This technique is complementary to metal-ligand studies performed by coupled electrochemical approaches and Raman spectroscopies in this spectral range.…”