Quinones in the A1 binding site in photosystem I studied using time-resolved FTIR difference spectroscopy

Abstract

Time-resolved step-scan FTIR difference spectroscopy at low temperature (77 K) has been used to study photosystem I particles with phylloquinone (2-methyl-3-phytyl-1,4-naphthaquinone) and menadione (2-methyl-1,4-naphthaquinone) incorporated into the A binding site. By subtracting spectra for PSI with phylloquinone incorporated from spectra for PSI with menadione incorporated a (menadione - phylloquinone) double difference spectrum was constructed. In the double difference spectrum bands associated with protein vibrational modes effectively cancel, and the bands in the spectrum are primarily associated with the neutral and reduced states of the two quinones in the A binding site. To aid in the assignment of bands in the experimental double difference spectrum, a double difference spectrum was calculated using three-layer ONIOM methods. The calculated and experimental spectra agree well, allowing unambiguous band assignments to be made. The ONIOM calculations show that both quinones in the A binding site are similarly oriented, with only a single hydrogen bond between the C=O quinone carbonyl group and the backbone NH group of a leucine residue. For the semi-quinone species, but not for the neutral species, this hydrogen bond appears to be very strong. Finally, we have for the first time been able to unmask and identify infrared difference bands associated with neutral naphthoquinone species occupying the A binding site in PSI.

Resolving the Impact of Hydrogen Bonding on the Phylloquinone Cofactor through Two-Dimensional Infrared Spectroscopy

“…When CO4 is hydrogen bonded, the bright CO4 transition is red-shifted to a frequency of 1651 cm –1 , and the bright CO1 transition has a frequency of 1669 cm –1 . The red shift in the frequency of the hydrogen bonded carbonyl unit is consistent with previous experimental and theoretical work focusing on carbonyl vibrational probes. , In addition, the observations are consistent with previous work focusing on assigning difference spectra of PhQ in the A 1 binding pocket of PSI, where two peaks are observed for the decoupled carbonyl modes of PhQ and the hydrogen bonded mode is red-shifted. , …”

“…These studies focused on characterizing the impact of a single hydrogen bond to a carbonyl unit of PhQ, where the local protein environment determined the geometry of the hydrogen bond. Based on the previous studies, the shoulder at 1650–1654 cm –1 would be consistent with hydrogen bonded PhQ, where the hydrogen bonding of the CO4 unit of PhQ in the A 1 binding pocket leads to a splitting of the carbonyl vibrational frequencies and an additional red-shifted transition. − In solution, the oxygen atom of ketone-like carbonyl groups can form up to 2 hydrogen bonds. , Thus, unlike in the protein binding environment, PhQ can form a total of 4 possible hydrogen bonding interactions, with 2 interactions per carbonyl group which might alter the modes of PhQ in slightly different ways. To fully characterize and assign the FTIR spectrum of PhQ in hexanol, we first determined the relative population of the different hydrogen bonding species through MD simulations and then performed DFT calculations on the most prominent hydrogen bonding species to extract their associated vibrational frequencies.…”

“…High-resolution X-ray crystallographic studies at cryogenic temperatures have identified π-stacking interactions between the naphthoquinone headgroup of PhQ and a tryptophan residue of the protein pocket, as well as an asymmetric hydrogen bonding interaction between the backbone NH group of a leucine side chain and a single carbonyl group of each PhQ molecule. , More recently, room temperature XFEL crystallographic studies were performed on PSI complexes, shedding light as to how slight structural differences in the π-stacking and hydrogen bonding interactions to the PhQ cofactors could lead to changes in their redox properties across the A and B branches of the reaction center . Additionally, spin-correlation techniques such as EPR − as well as experimental and theoretical FTIR difference spectra − have been used to probe both the neutral and anionic forms of PhQ in PSI, where the results were interpreted in the context of local protein interactions. Although there is much previous work studying the protein–cofactor interactions in the A 1 binding site, there have been limited investigations characterizing how these interactions evolve on the ultrafast time scales relevant to charge separation.…”

“…To accurately extract information on the local environment from 2DIR measurements, the vibrational probe must be well-characterized such that the changes in dynamics and spectral properties can be definitively ascribed to changes in the local environmentrather than being intrinsic to the molecule. In particular, carbonyl groups are a set of well-characterized vibrational reporters which have a remarkable sensitivity to their environment. − Considering the structure of PhQ (Figure ), the two carbonyl groups (CO1 and CO4) have further been demonstrated to serve as vibrational probes in previous FTIR-based studies of the A 1 binding site in PSI, ,,, where the asymmetric hydrogen bonding between CO4 and an amino acid side chain leads to a large splitting in the vibrational frequencies associated with the carbonyl stretching modes. Previously, 2DIR measurements have been used to study different quinone headgroups, showing that 2DIR spectroscopy can effectively characterize the energetics of the quinone carbonyl groups .…”

Room temperature XFEL crystallography reveals asymmetry in the vicinity of the two phylloquinones in photosystem I

“…In a FTIR study, it is also suggested that the redox potential difference between the quinones may stem from a stronger H-bonding of PhQ A — vs PhQ B — 62 . The above discussed rotation of PsaB-Phe669 to the reduced state stabilizing T-stacking, may weaken the PhQ B H-bond at the opposite side.…”