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1. Application of the Triangular Spatial Relationship Algorithm in Representing and Quantifying Conformational Changes in Chlorophylls and Protein Local Environments

   https://doi.org/10.3390/photochem5010008  

   Milon, Tarikul I; Orthi, Khairum H; Rauniyar, Krishna; Renfrow, Rhen M; Gallo, August A; Xu, Wu (March 2025, Photochem)

   Chemically identical chlorophyll (Chl) molecules undergo conformational changes when they are embedded in a protein matrix. The conformational changes will modulate their absorption spectra to meet the need for programmed excitation energy transfer or electron transfer. To interpret spectroscopic data using the knowledge of pigment–protein interactions requires a single pigment embedded in one polypeptide matrix. Unfortunately, most of the known photosynthetic systems contain a set of multiple pigments in each protein subunit. This makes it complicated to interpret spectroscopic data using structural data due to the potential overlapping spectra of two or more pigments. Chl–protein interactions have not been systematically studied to answer three fundamental questions: (i) What are the structural characteristics and commonly shared substructures of different types of Chl molecules (e.g., Chl a, b, c, d, and f)? (ii) How many structural groups can Chl molecules be divided into and how are different structural groups influenced by their surrounding environments? (iii) What are the structural characteristics of pigment surrounding environments? Having no clear answers to the unresolved questions is probably due to a lack of computational methods for quantifying conformational changes in individual Chls and individual surrounding amino acids. The first version of the Triangular Spatial Relationship (TSR)-based method was developed for comparing protein 3D structures. The input data for the TSR-based method are experimentally determined 3D structures from the Protein Data Bank (PDB). In this study, we take advantage of the 3D structures of Chl-binding proteins deposited in the PDB and the TSR-based method to systematically investigate the 3D structures of various types of Chls and their protein environments. The key contributions of this study can be summarized as follows: (i) Specific structural characteristics of Chl d and f were identified and are defined using the TSR keys. (ii) Two and three clusters were found for various types of Chls and Chls a, respectively. The signature structures for distinguishing their corresponding two and three clusters were identified. (iii) Histidine residues were used as an example for revealing structural characteristics of Chl-binding sites. This study provides evidence for the three unresolved questions and builds a structural foundation through quantifying Chl conformations as well as structures of their embedded protein environments for future mechanistic understanding of relationships between Chl–protein interactions and their corresponding spectroscopic data. 

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2. Impact of Peripheral Hydrogen Bond on Electronic Properties of the Primary Acceptor Chlorophyll in the Reaction Center of Photosystem I

   https://doi.org/10.3390/ijms25094815  

   Luo, Lujun; Martin, Antoine P; Tandoh, Elijah K; Chistoserdov, Andrei; Slipchenko, Lyudmila V; Savikhin, Sergei; Xu, Wu (May 2024, International Journal of Molecular Sciences)

   Photosystem I (PS I) is a photosynthetic pigment–protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A−1, A0, A1, respectively), converging in a single iron–sulfur complex Fx. While there is a consensus that the ultimate electron donor–acceptor pair is P700+A0−, the involvement of A−1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr → Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr → Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role. 

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3. Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

   https://doi.org/10.1038/s41467-022-32727-w  

   Zhang, Linyue; King, Edward; Black, William B.; Heckmann, Christian M.; Wolder, Allison; Cui, Youtian; Nicklen, Francis; Siegel, Justin B.; Luo, Ray; Paul, Caroline E.; et al (August 2022, Nature Communications)

   Abstract Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase inEscherichia colito exclusively rely on the reduced NMN⁺(NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺. 

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4. Encapsulation of tricopper cluster in a synthetic cryptand enables facile redox processes from Cu ^I Cu ^I Cu ^I to Cu ^II Cu ^II Cu ^II states

   https://doi.org/10.1039/D0SC05441K  

   Zhang, Weiyao; Moore, Curtis E.; Zhang, Shiyu (March 2021, Chemical Science)

   null (Ed.)

   One-pot reaction of tris(2-aminoethyl)amine (TREN), [Cu I (MeCN) 4 ]PF 6 , and paraformaldehyde affords a mixed-valent [ TREN4 Cu II Cu I Cu I (μ 3 -OH)](PF 6 ) 3 complex. The macrocyclic azacryptand TREN4 contains four TREN motifs, three of which provide a bowl-shape binding pocket for the [Cu 3 (μ 3 -OH)] 3+ core. The fourth TREN caps on top of the tricopper cluster to form a cryptand, imposing conformational constraints and preventing solvent interaction. Contrasting the limited redox capability of synthetic tricopper complexes reported so far, [ TREN4 Cu II Cu I Cu I (μ 3 -OH)](PF 6 ) 3 exhibits several reversible single-electron redox events. The distinct electrochemical behaviors of [ TREN4 Cu II Cu I Cu I (μ 3 -OH)](PF 6 ) 3 and its solvent-exposed analog [ TREN3 Cu II Cu II Cu II (μ 3 -O)](PF 6 ) 4 suggest that isolation of tricopper core in a cryptand enables facile electron transfer, allowing potential application of synthetic tricopper complexes as redox catalysts. Indeed, the fully reduced [ TREN4 Cu I Cu I Cu I (μ 3 -OH)](PF 6 ) 2 can reduce O 2 under acidic conditions. The geometric constraints provided by the cryptand are reminiscent of Nature's multicopper oxidases (MCOs). For the first time, a synthetic tricopper cluster was isolated and fully characterized at Cu I Cu I Cu I ( 4a ), Cu II Cu I Cu I ( 4b ), and Cu II Cu II Cu I ( 4c ) states, providing structural and spectroscopic models for many intermediates in MCOs. Fast electron transfer rates (10 5 to 10 6 M −1 s −1 ) were observed for both Cu I Cu I Cu I /Cu II Cu I Cu I and Cu II Cu I Cu I /Cu II Cu II Cu I redox couples, approaching the rapid electron transfer rates of copper sites in MCO. 

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5. Structural insights into melatonin receptors

   https://doi.org/10.1111/febs.15128  

   Stauch, Benjamin; Johansson, Linda C.; Cherezov, Vadim (November 2019, The FEBS Journal)

   The long‐anticipated high‐resolution structures of the human melatonin G protein‐coupled receptors MT₁and MT₂, involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti‐insomnia and antidepression drugs using X‐ray free‐electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane‐buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT₂from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT₂structure allowed accurate mapping of type 2 diabetes‐related single‐nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein–membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT₁and MT₂with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next‐generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects. 

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