Band assignment for quinone in the A 1 binding site

Figure 2.9 Calculated and experimental DS of PhQ- and 2MNQ- (A) Experimental FTIR DDS obtained by subtracting A1-/A1 spectrum of 2MNQ from that of PhQ (B) Calculated DS of PhQ- and 2MNQ- in the gas phase by using DFT methods (Model 1); (C) in the presence of a truncated Leu residue by using DFT methods (Model 2); and (D) by using ONIOM method (Model 3). Frequencies in B, C and D were scaled by 0.973/0.973/0.96, respectively.

The calculated and experimental (2MNQ-PhQ) DDS expanded in anion quinone spectral region (1550-1390 cm-1) were compared and shown in Figure 2.9. Figure 2.9A shows the

experimental (2MNQ-PhQ) FTIR DDS. Figure 2.9 B shows the calculated (2MNQ-PhQ) DDS obtained using Model 1. Figure 2.9C shows the calculated (2MNQ-PhQ) DDS for Model 2, a PhQ molecule in the presence of a truncated Leu residue. The ONIOM calculated DS (2MNQ- PhQ) DDS is given in Figure 2.9D. In the anion quinone spectra region, bands of 2MNQ- are positive, while bands of PhQ- are negative.

One prominent observation for the experimental and calculated DDS is the difference band at 1494 (-) /1504(+) cm-1. Isotope edited FTIR DS have suggested that the band at 1494 cm- 1

in A1-/A1 FTIR DS is due to a C ⃛O mode of PhQ- and downshifts 12 cm-1, 41 cm-1and 14 cm-1 upon deuteration, 13C and 18O isotope labeling previously [82, 87]. The experimental (2MNQ- PhQ) FTIR DDS suggests that the PhQ- band at 1494 cm-1 upshifts 10 cm-1 to 1504 cm-1, upon replacing PhQ with 2MNQ in the A1 binding site. This result is in good agreement with

calculated spectra from Model 1-3. In DFT based vibrational frequency calculations (Model 1 and 2), the band at 1494 cm-1 was assigned to antisymmetric stretching of two C⃛O mode of PhQ-. However, in Model 3, the band at 1494 cm-1 is mainly due to the stretching of C1⃛O mode of PhQ- and upshifts ~15 cm-1 for 2MNQ- when the phytyl tail is replaced by a hydrogen atom.

Another very clear feature in the DDS is the difference band at 1415/1427 cm-1, indicating a band of PhQ- at 1415 cm-1 with the corresponding band of 2MNQ at 1427 cm-1.

In Model 1 and 2, the 1415 cm-1 band in A1-/A1 FTIR DS is suggested to be

predominantly due to a C-H bending vibrations and weakly coupled to aromatic C-C stretching and antisymmetric C⃛O stretching vibration. The experimental DDS indicates that this mode

upshifts 12 cm-1 to 1427 cm-1 for 2MNQ-. Upon replacement of the phytyl chain of PhQ- with a hydrogen, Model 1 suggests that the band at 1415 cm-1downshifts 4 cm-1, while Model 2 suggest that the band at 1415 cm-1upshifts 7 cm-1. However, the intensity of the difference band at 1439(+)/1432(-) cm-1in the calculated spectrum obtained from Model 2 is too small. In Model 3, the band at 1409 cm-1 is mainly assigned to the C4⃛O mode stretching coupled to C-H bending of the truncated tail of PhQ- and upshifts 9 cm-1 for the corresponding mode of 2MNQ- at 1418 cm-1. Therefore, the antisymmetric H-bond should be involved in the model for vibrational frequency calculations, and both intensities and positions of difference bands in the calculated

spectrum from Model 3 are closed to experimental results. In the calculated spectrum from Model 3, the difference band at 1427(+)/1415(-) cm-1, which is due to C4⃛O mode of reduced quinone, downshifts ~80 cm-1 compared to C1⃛O mode of reduced quinone. The ONIOM calculations suggest that asymmetric H-bond uncouples the two C⃛O modes of PhQ-.

Another feature in the experimental DDS is the difference band at 1469/1477 cm-1, perhaps indicating a band of PhQ- at 1469 cm-1 with a corresponding band in 2MNQ at 1477 cm- 1

. The ONIOM calculation (Model 3) suggests the band at 1484 cm-1 is due to the stretching of the C4⃛Ogroup coupling to C-H bending of the methyl group at 2-position, with a

corresponding band in 2MNQ- at 1469 cm-1.

Figure 2.10 Calculated and experimental DS of PhQ and 2MNQ (A) Experimental FTIR DDS obtained by subtracting A1-/A1 spectrum of 2MNQ from that of PhQ (B) Calculated DS of PhQ and 2MNQ in the gas phase by using DFT methods (Model 1); (C) in the presence of a truncated Leu residue by using DFT methods (Model 2); and (D) by using ONIOM method (Model 3). Frequencies in B, C and D were scaled by 0.965/0.965/0.952, respectively.

Experimental (2MNQ-PhQ) DDS in the region of 1700~1550 cm-1 is shown in the Figure 2.10. Calculated DS relative to experiments with different models are also shown in Figure 2.10. Figure 2.10 B shows the DS of PhQ and 2MNQ in gas phase by using DFT methods. Figure 2.10C shows the calculated DS of PhQ and 2MNQ in the presence of a truncated Leu residue. The ONIOM calculated DS of PhQ and 2MNQ is given in Figure 2.10D. In the neutral region of DDS, bands of 2MNQ are negative, while bands of PhQare positive. The neutral quinone spectra is complicated and poorly understood.

A very clear feature in the experimental DDS from 1800~1550 cm-1 is the difference band at 1663/1656 cm-1, indicating a band of PhQat 1656 cm-1 with a corresponding band in 2MNQ at 1663 cm-1. A C=O absorption band of PhQ in solvent is observed at ~1662 cm-1. More specifically, the normal mode, which gives rise to most of the intensity of the 1662 cm-1, band is an asymmetric stretching vibration of both C=O groups. The frequency of this normal mode upshifts to 1666 cm-1 when the phytyl chain is replaced by a hydrogen atom (Figure 2.6).

FTIR spectroscopy studies also suggested that the negative band near 1655 cm-1 in the trimeric A1-/A1 FTIR DS is little impacted by deuteration and downshifts ~26 cm-1 upon 18O labeling [82, 87]. Therefore, a frequency of 1655 cm-1 is due to a PhQ C=O mode that is free from H-bonding. ONIOM calculation of PhQ in the neutral state also shows that the band at 1656 cm-1 is due to the C1=O stretching of PhQ, which is free from H-bonding. In addition, ONIOM calculation suggests that frequency of 2MNQ C1=O stretching in the A1 binding site is 7 cm-1 higher than that of PhQ. Therefore, the ONIOM calculations suggest that the difference band at 1663/1656 cm-1 in the (2MNQ-PhQ) DDS is due to the C1=O stretching of 2MNQ/PhQ.

ONIOM calculations of PhQ and 2MNQ show that a negative band at 1638 cm-1 in the experimental (2MNQ-PhQ) DDS is not due to quinone. In the previous studies, it was found the

difference band at 1643/1634 cm-1 in the A1-/A1 FTIR DS downshifted 8~10 cm-1 upon deuteration, suggesting it could be due to amide I mode [82].

In summary, in the anion quinone region of (2MNO-PhQ) DDS, the bands of 2MNQ at 1504 and 1428 cm-1 corresponds to the bands of PhQ at 1495 and 1415 cm-1.The comparison of these bands is taken to further support the C=O character previously assigned to the 1654, 1495, and 1415 cm-l bands of PhQ in the A1 binding site. In the calculated spectrum of ONIOM calculations, the vibrational frequency of C4=O group of PhQ- downshifts ~80 cm-1 compared to the vibrational frequency of C1=O group. This observation agrees with the notion of a strong H- bonding to semiquinone derived from ENDOR studies of A1-. The band at 1656 cm-1 in the (2MNQ-PhQ) FTIR DDS is due to PhQ, with corresponding mode of 2MNQ at 1663 cm-1. The ONIOM calculation also predict that the antisymmetric H-bond also uncouples the two C=O modes of neutral PhQ, and C4=O group of PhQ downshifts ~60 cm-1 compared to the vibrational frequency of C1=O group. The close analogy between the DDS calculated for RCs reconstituted either with PhQ or with 2MNQ shows that the phytyl chain of PhQ imparts no specific

constraint on the geometry of the menaquinone head group in its binding site for both the neutral and reduced state.