Conformational Heterogeneity of the Bacteriopheophytin Electron Acceptor H_A in Reaction Centers from Rhodopseudomonas viridis Revealed by Fourier Transform Infrared Spectroscopy and Site-Directed Mutagenesis

Abstract

The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.

Mutation-induced perturbation of the special pair P840 in the homodimeric reaction center in green sulfur bacteria

“…The strongest peak at 1697 cm −1 can hence be attributed to the overlap of the H-bonded C=O band downshifted by 10 cm −1 from 1707 cm −1 and the non-H-bonded band that remained at 1695 cm −1 . A similar example of the coexistence of H-bonded and non-H-bonded forms has been observed in the 13 2 -ester C=O of the bacteriopheophytin electron acceptor in PbRC 49 . The equilibrium of 2 H-bonded forms at the keto C=O in P840 + seems absent in neutral P840 of the V689C RC, because the original WT band at 1682 cm −1 , which reflects a non-H-bond interaction, did not remain in the spectrum of the V689C RC.…”

Ubiquinone Binding, Ubiquinone Exclusion, and Detailed Cofactor Conformation in a Mutant Bacterial Reaction Center

“…sphaeroides reaction centers, termed H L1 -and H L2 - (58,59). The relevance of these conformations to relaxation processes and the energetics of primary electron transfer have been discussed (58)(59)(60). It was suggested that the H L1 -state, with φ Ac ) 45°or 315°(i.e., -45°), and the H L2 -state, with φ Ac ) 135°or 225°(i.e.…”

Local and Global Electric Field Asymmetry in Photosynthetic Reaction Centers

“…In particular, numerous studies focused on the assignment of hydrogen bonds to the 9-keto and 2-acetyl groups because they are part of the delocalized π-system and any change in H-bonding is expected to affect the redox properties of the chromophores and hence, ET rates . Light-minus-dark FTIR difference spectroscopy, pioneered by Breton et al , allows for the assignment of the carbonyl frequencies of the chromophores in the part of the RC, where ET occurs (P L , B L , H L , Q A , and Q B ). ,, Vibrational frequencies of the chromophores in the inactive M-branch (P M , B M , and H M ) cannot be assigned using this method. In contrast, resonance Raman techniques enable assignment of the carbonyl modes of all chromophores because the ground electronic absorption spectrum shows well-enough resolved bands for all chromophores at low temperature allowing for selective enhancement of vibrational modes of each individual chromophore. , …”

“…In particular, numerous studies focused on the assignment of hydrogen bonds to the 9-keto and 2-acetyl groups because they are part of the delocalized π-system and any change in H-bonding is expected to affect the redox properties of the chromophores and hence, ET rates. 44 Light-minus-dark FTIR difference spectroscopy, pioneered by Breton et al 21,45 allows for the assignment of the carbonyl frequencies of the chromophores in the part of the RC, where ET occurs (P L , B L , H L , Q A , and Q B ). 5,21,45 Vibrational frequencies of the chromophores in the inactive M-branch (P M , B M , and H M ) cannot be assigned using this method.…”