HYSCORE and QM/MM Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Remote Nitrogens.

Secondary coordination sphere (SCS) interactions have been shown to play important roles in tuning reduction potentials and electron transfer (ET) properties of the Type 1 copper proteins, but the precise roles of these interactions are not fully understood. In this work, we examined the influence of F114P, F114N, and N47S mutations in the SCS on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of (i) histidine remote Nε nitrogens and (ii) the amide Np using the two-dimensional (2D) pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) combined with quantum mechanics/molecular mechanics (QM/MM) and DLPNO-CCSD calculations. Our data show that some components of hyperfine tensor and isotropic coupling in N47SAz and F114PAz (but not F114NAz) deviate by up to ∼±20% from WTAz, indicating that these mutations significantly influence the spin density distribution between the CuII site and coordinating ligands. Furthermore, our calculations support the assignment of Np to the backbone amide of residue 47 (both in Asn and Ser variants). Since the spin density distributions play an important role in tuning the covalency of the Cu-Scys bond of Type 1 copper center that has been shown to be crucial in controlling the reduction potentials, this study provides additional insights into the electron spin factor in tuning the reduction potentials and ET properties.

A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model of lytic polysaccharide monooxygenase.

The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His)2 with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV-Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.

Influence of Polar Mutations on the Electronic and Structural Properties of QA-· in Bacterial Reaction Centers.

Reaction centers from Rhodobacter sphaeroides with residue M265 mutated from isoleucine to threonine, serine, and asparagine (M265IT, M265IS, and M265IN, respectively) in the QA-· state are studied by high-resolution electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance spectroscopy methods to investigate the structural characteristics of these mutants influencing the redox properties of the QA site. All three mutants decrease the redox midpoint potential (Em) of QA by ∼0.1 V, yet the mechanism for this drop in Em is unclear. In this work, we examine (i) the hydrogen bonding interactions between QA-· and residues histidine M219 and alanine M260, (ii) the electron spin density distribution of the semiquinone, and (iii) the orientations of the ubiquinone methoxy substituents. 13C measurements show no significant contribution of methoxy dihedral angles to the observed decrease in Em for the QA mutants. Instead, 14N three-pulse ESEEM data suggest that electrostatic or hydrogen bond formation between the mutated M265 side chain and His-M219 Nδ may be involved in the observed lowering of the QA midpoint potential. For mutant M265IN, analysis of the proton hyperfine couplings reveals a weakened hydrogen bond network, resulting in an altered QA-· spin density distribution. The magnetic resonance study presented here is most consistent with an electrostatic or structural perturbation of the His-M219 Nδ hydrogen bond in these mutants as a mechanism for the ∼0.1 V decrease in QA Em.

Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships.

A set of C43(DE3) and BL21(DE3) Escherichia coli host strains that are auxotrophic for various amino acids is briefly reviewed. These strains require the addition of a defined set of one or more amino acids in the growth medium, and have been specifically designed for overproduction of membrane or water-soluble proteins selectively labelled with stable isotopes, such as 2H, 13C and 15N. The strains described here are available for use and have been deposited into public strain banks. Although they cannot fully eliminate the possibility of isotope dilution and mixing, metabolic scrambling of the different amino acid types can be minimized through a careful consideration of the bacterial metabolic pathways. The use of a suitable auxotrophic expression host strain with an appropriately isotopically labelled growth medium ensures high levels of isotope labelling efficiency as well as selectivity for providing deeper insight into protein structure-function relationships.

A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold.

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

The Ubiquinol Binding Site of Cytochrome bo3 from Escherichia coli Accommodates Menaquinone and Stabilizes a Functional Menasemiquinone.

Cytochrome bo3, one of three terminal oxygen reductases in the aerobic respiratory chain of Escherichia coli, has been well characterized as a ubiquinol oxidase. The ability of cytochrome bo3 to catalyze the two-electron oxidation of ubiquinol-8 requires the enzyme to stabilize the one-electron oxidized ubisemiquinone species that is a transient intermediate in the reaction. Cytochrome bo3 has been shown recently to also utilize demethylmenaquinol-8 as a substrate that, along with menaquinol-8, replaces ubiquinol-8 when E. coli is grown under microaerobic or anaerobic conditions. In this work, we show that its steady-state turnover with 2,3-dimethyl-1,4-naphthoquinol, a water-soluble menaquinol analogue, is just as efficient as with ubiquinol-1. Using pulsed electron paramagnetic resonance spectroscopy, we demonstrate that the same residues in cytochrome bo3 that stabilize the semiquinone state of ubiquinone also stabilize the semiquinone state of menaquinone, with the hydrogen bond strengths and the distribution of unpaired spin density accommodated for the different substrate. Catalytic function with menaquinol is more tolerant of mutations at the active site than with ubiquinol. A mutation of one of the stabilizing residues (R71H in subunit I) that eliminates the ubiquinol oxidase activity of cytochrome bo3 does not abolish activity with soluble menaquinol analogues.

HYSCORE Insights into the Distribution of the Unpaired Spin Density in an Engineered CuA Site in Azurin and Its His120Gly Variant.

A comparative study of the 1H and 14N hyperfine interactions between the CuA site in an engineered CuA center in azurin (WT-CuAAz) and its His120Gly variant (H120G-CuAAz) using the two-dimensional ESEEM technique, HYSCORE, is reported. HYSCORE spectroscopy has clarified conflicting results in previous electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies and found clear differences between the two CuA azurins. Specifically, a hyperfine coupling AN⊥ of 15.3 MHz was determined for the first time from the frequencies of double-quantum transitions of 14N histidine nitrogens coordinated to CuA in WT-CuAAz. In contrast, such coupling was not observed in the spectra of H120G-CuAAz, indicating at least a several megahertz increase in AN⊥ for the coordinated nitrogen in this variant. In addition, 14N HYSCORE spectra of WT-CuAAz show interaction with only one type of weakly coupled nitrogen assigned to the remote Nε atom of coordinated imidazole residues based on the quadrupole coupling constant ( e2 Qq/4 h) of ∼0.4 MHz. The spectrum of H120G-CuAAz resolves additional features typical for backbone peptide nitrogens with larger e2 Qq/4 h values of ∼0.7 MHz. Hyperfine couplings with these nitrogens vary between ∼0.4 and 0.7 MHz. In addition, the two resolved cross-peaks from Cß protons in H120G-CuAAz display only ∼1 MHz shifts relative to the corresponding peaks in WT-CuAAz. These new findings have provided the first experimental evidence of the previous density functional theory analysis that predicted changes in the delocalized electron spin population of ∼0.02-0.03 (i.e., ∼10%) on copper and sulfur atoms of the CuA center in H120 variants relative to WT-CuAAz and resolved contradicting results between EPR and ENDOR studies of the valence distribution in CuAAz and its variants.

Convolutional Neural Network Analysis of Two-Dimensional Hyperfine Sublevel Correlation Electron Paramagnetic Resonance Spectra.

A machine learning approach is presented for analyzing complex two-dimensional hyperfine sublevel correlation electron paramagnetic resonance (HYSCORE EPR) spectra with the proficiency of an expert spectroscopist. The computer vision algorithm requires no training on experimental data; rather, all of the spin physics required to interpret the spectra are learned from simulations alone. This approach is therefore applicable even when insufficient experimental data exist to train the algorithm. The neural network is demonstrated to be capable of utilizing the full information content of two-dimensional 14N HYSCORE spectra to predict the magnetic coupling parameters and their underlying probability distributions that were previously inaccessible. The predicted hyperfine ( a, T) and 14N quadrupole ( K, η) coupling constants deviate from the previous manual analyses of the experimental spectra on average by 0.11 MHz, 0.09 MHz, 0.19 MHz, and 0.09, respectively.

g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance.

Interpretation of magnetic resonance data in the context of structural and chemical biology requires prior knowledge of the g-tensor directions for paramagnetic metallo-cofactors with respect to the protein structural frame. Access to this information is often limited by the strict requirement of suitable protein crystals for single-crystal electron paramagnetic resonance (EPR) measurements or the reliance on protons (with ambiguous locations in crystal structures) near the paramagnetic metal site. Here we develop a novel pulsed EPR approach with selective 13Cß-cysteine labeling of model [2Fe-2S] proteins to help bypass these problems. Analysis of the 13Cß-cysteine hyperfine tensors reproduces the g-tensor of the Pseudomonas putida ISC-like [2Fe-2S] ferredoxin (FdxB). Its application to the hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus, for which the single-crystal EPR approach was not feasible, supports the best-fit g x-, g z-, and g y-tensor directions of the reduced cluster as nearly along Fe-Fe, S-S, and the cluster plane normal, respectively. These approximate principal directions of the reduced ARF g-tensor, explored by 13C pulsed EPR, are less skewed from the cluster molecular axes and are largely consistent with those previously determined by single-crystal EPR for the cytochrome bc1-associated, reduced Rieske [2Fe-2S] center. This suggests the approximate g-tensor directions are conserved across the phylogenetically and functionally divergent Rieske-type [2Fe-2S] proteins.

Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in Nitrogen Hydrogen Bond Donors of Semiquinone Intermediates.

Hydrogen bonding between semiquinone (SQ) intermediates and side-chain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the 15N HYSCORE spectra not reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Q-sites of wild-type and mutant D75H cytochrome bo3 ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the a-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width, Δ a, and parameter, δ = |2 a + T| - 4ν15N. α-strain provides a direct measure of the structural dynamics and heterogeneity of the O···H···N bond in the SQ systems.

Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by 13Cß-Cysteine Labeling.

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4-n (n = 0, 1, 2) cluster by selective cysteine-13Cß isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cß hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

Determination of the Complete Spin Density Distribution in 13C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory.

Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically 13C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary QA and secondary QB ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides 13C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected 13C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and pπ-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.

Pulsed Electron Paramagnetic Resonance Insights into the Ligand Environment of Copper in Drosophila Lysyl Oxidase.

Lysyl oxidase (LOX) is a copper amine oxidase that cross-links collagens and elastin in connective tissue and plays an important role in fibrosis, cancer development, and formation of the "metastatic niche". Despite its important biological functions, the structure of human LOX remains unknown (unlike that of an unrelated LOX, from Pichia pastoris). Here, we expressed active LOX from Drosophila melanogaster, DmLOXL1, a close homologue of human LOX, and characterized it by MS, UV-vis, activity, and inhibition assays. We then used bioinformatics, electron paramagnetic resonance, electron spin-echo envelope modulation, and hyperfine sublevel-correlation (HYSCORE) spectroscopies to probe Cu-ligand bonding finding direct evidence for pH-dependent Cu-His interactions. At pH = 9.3, the spectroscopic data indicated primarily a single His bound to Cu, but at pH = 7.5, there was evidence for a ∼ 1:1 mixture of species containing 1 and 3 His ligands. We then used HYSCORE to probe possible interactions between the LOX inhibitor BAPN (ß-aminopropionitrile; 1-[13C15N]cyano-2-aminoethane) and the copper center-finding none. Overall, the results are of interest since they provide new spectroscopic information about the nature of the catalytic site in LOX, an important anticancer drug target.

The Q-Cycle Mechanism of the bc1 Complex: A Biologist's Perspective on Atomistic Studies.

The Q-cycle mechanism of the bc1 complex is now well enough understood to allow application of advanced computational approaches to the study of atomistic processes. In addition to the main features of the mechanism, these include control and gating of the bifurcated reaction at the Qo-site, through which generation of damaging reactive oxygen species is minimized. We report a new molecular dynamics model of the Rhodobacter sphaeroides bc1 complex implemented in a native membrane, and constructed so as to eliminate blemishes apparent in earlier Rhodobacter models. Unconstrained MD simulations after equilibration with ubiquinol and ubiquinone respectively at Qo- and Qi-sites show that substrate binding configurations at both sites are different in important details from earlier models. We also demonstrate a new Qo-site intermediate, formed in the sub-ms time range, in which semiquinone remains complexed with the reduced iron sulfur protein. We discuss this, and a spring-loaded mechanism for modulating interactions of the iron sulfur protein with occupants of the Qo-site, in the context of control and gating roles. Such atomistic features of the mechanism can usefully be explored through simulation, but we stress the importance of constraints from physical chemistry and biology, both in setting up a simulation and in interpreting results.

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz' = 11.8 MHz, whereas for H2, Tz' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

Regulation of the primary quinone binding conformation by the H subunit in reaction centers from Rhodobacter sphaeroides.

Unlike photosystem II (PSII) in higher plants, bacterial photosynthetic reaction centers (bRCs) from Proteobacteria have an additional peripheral membrane subunit "H". The H subunit is necessary for photosynthetic growth, but can be removed chemically in vitro. The remaining LM dimer retains its activity to perform light-induced charge separation. Here we investigate the influence of the H subunit on interactions between the primary semiquinone and the protein matrix, using a combination of site-specific isotope labeling, pulsed electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. The data reveal substantially weaker binding interactions between the primary semiquinone and the LM dimer than observed for the intact bRC; the amount of electron spin transferred to the nitrogen hydrogen bond donors is significantly reduced, the methoxy groups are more free to rotate, and the spectra indicate a heterogeneous mixture of bound semiquinone states. These results are consistent with a loosening of the primary quinone binding pocket in the absence of the H subunit.

Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis.

Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.

Hydrogen bond network around the semiquinone of the secondary quinone acceptor Q(B) in bacterial photosynthetic reaction centers.

By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the QB site semiquinone (SQB) was determined. The development of such a technique is crucial toward an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQB hydrogen bond network was investigated with Q- (∼34 GHz) and X-band (∼9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQB g-tensor reference frame provided the principal values and directions of the H-bond proton hyperfine tensors. Three protons were detected, one with an anisotropic tensor component, T = 4.6 MHz, assigned to the histidine NδH of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQB g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQB used in previous DFT work is in strong agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.

Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides.

Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the interactions of the protein with the quinones in the Q(A) and Q(B) sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the Q(B) site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the Q(A) and Q(B) sites. Disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the Q(A)⁻Q(B)⁻ biradical and competitive binding assays.