Pub Date : 2025-12-18DOI: 10.1016/j.jinorgbio.2025.113191
Andrew J. Smith , Cadmus D. Chen , Bochu Wang , Xiao Huang , Chenfengtao Zheng , Youhyun Nam , David N. Beratan
Charge transfer and transport are fundamental to chemistry and biochemistry. Since the 1950s, theorists and experimentalists have advanced the field to develop a molecular-level understanding of single-electron/one-step electron transfer, and ongoing studies explore multi-step, multi-electron, and multi-particle transfer and transport. This review highlights studies from our group on electron transfer beyond the one-electron/one-step regime. In particular, we review progress on electron bifurcation reactions, ultra-long range hopping transport in cable bacteria and bacterial nanowires, oxidative damage pathways, and charge transport pathways in nucleic acids. We highlight emerging themes and mechanisms across biological structures, including the roles being discovered for electron-electron correlations, rapid long-range multi-step biological electron transport, unconventional charge transport pathways, and competing charge transfer mechanisms.
{"title":"From single step hops to emergent biological phenomena","authors":"Andrew J. Smith , Cadmus D. Chen , Bochu Wang , Xiao Huang , Chenfengtao Zheng , Youhyun Nam , David N. Beratan","doi":"10.1016/j.jinorgbio.2025.113191","DOIUrl":"10.1016/j.jinorgbio.2025.113191","url":null,"abstract":"<div><div>Charge transfer and transport are fundamental to chemistry and biochemistry. Since the 1950s, theorists and experimentalists have advanced the field to develop a molecular-level understanding of single-electron/one-step electron transfer, and ongoing studies explore multi-step, multi-electron, and multi-particle transfer and transport. This review highlights studies from our group on electron transfer beyond the one-electron/one-step regime. In particular, we review progress on electron bifurcation reactions, ultra-long range hopping transport in cable bacteria and bacterial nanowires, oxidative damage pathways, and charge transport pathways in nucleic acids. We highlight emerging themes and mechanisms across biological structures, including the roles being discovered for electron-electron correlations, rapid long-range multi-step biological electron transport, unconventional charge transport pathways, and competing charge transfer mechanisms.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"276 ","pages":"Article 113191"},"PeriodicalIF":3.2,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-18DOI: 10.1016/j.jinorgbio.2025.113199
Piotr J. Mak , Sandra Acebes , Victor Guallar , James R. Kincaid , Anabella Ivancich
We have fully characterized the high-valent intermediates formed during the catalytic cycle of lactoperoxidase (LPO), a heme-containing enzyme present in milk, saliva, tears and airways, playing an important role in mammalian antimicrobial defense. Our combined approach of multifrequency (9–285 GHz) Electron Paramagnetic Resonance (EPR) and resonance Raman (rR) spectroscopies allowed the identification of Tyr21 and Tyr193 as radical sites, having a concerted role with the heme iron and resulting in two distinct [Fe(IV) = O Tyr•] intermediates in LPO's catalytic cycle. The presence and strength of H-bonds to Tyr• assessed by the C − O stretching mode (v7a) and the gx component of the g-tensor detected in their rR and 285-GHz EPR spectra, respectively, induced a difference in pH-dependent stabilization of the [Fe(IV) = O Tyr21•] and [Fe(IV) = O Tyr193•] intermediates. The later resulted from single-step electron tunneling subsequent to the formation of the [Fe(IV) = O Por•+] species. A Trp radical contributing to the LPO 285-GHz EPR spectra, discern by the advantageous resolution of g-values, is proposed to facilitate the electron transfer to Tyr193. Our computational analysis (PELE) crucially identified a unique surface binding site for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), close to Tyr21 (shown to host the spin by QM/MM simulations), thus consistent with LPO Tyr• pH 5.6 detected in the rR and 285-GHz EPR spectra and the substrate oxidation previously shown by our UV–Vis stopped flow electronic absorption studies. The elucidation of the catalytic electron transfer pathways and the protein-based oxidizing sites is relevant to harnessing the concerted reactivity of heme and specific Tyr/Trp sites in natural biocatalysts.
我们已经充分表征了在乳酸过氧化物酶（LPO）催化循环中形成的高价中间体，LPO是一种含血红素的酶，存在于牛奶、唾液、眼泪和气道中，在哺乳动物的抗菌防御中起重要作用。我们的多频（9-285 GHz）电子顺磁共振（EPR）和共振拉曼（rR）光谱组合方法可以识别Tyr21和Tyr193为自由基位点，它们与血红素铁协同作用，并在LPO的催化循环中产生两种不同的[Fe(IV) = O Tyr•]中间体。通过C−O拉伸模式（v7a）评估的Tyr•氢键的存在和强度，以及在rR和285-GHz EPR光谱中检测到的g张量的gx分量，分别导致了[Fe(IV) = O Tyr21•]和[Fe(IV) = O Tyr193•]中间体的ph依赖稳定性的差异。后者是由[Fe(IV) = O Por•+]物质形成后的单步电子隧穿引起的。一个Trp自由基对LPO 285-GHz EPR谱有贡献，通过有利的g值分辨，被提出促进电子转移到Tyr193。我们的计算分析（PELE）至关重要地确定了2,2 ' -氮基-双（3-乙基苯并噻唑-6-磺酸）（ABTS）的独特表面结合位点，接近Tyr21（通过QM/MM模拟显示其承载自旋），因此与在rR和285-GHz EPR光谱中检测到的LPO Tyr•pH 5.6以及之前我们的UV-Vis停止流动电子吸收研究中显示的底物氧化一致。催化电子转移途径和基于蛋白质的氧化位点的阐明与利用天然生物催化剂中血红素和特异性Tyr/Trp位点的协同反应性有关。
{"title":"Lactoperoxidase: Identification of the two Tyr radical sites for the stabilization of the pH-dependent [Fe(IV) = O Tyr•] catalytic intermediates by resonance Raman and multifrequency Electron Paramagnetic Resonance spectroscopies","authors":"Piotr J. Mak , Sandra Acebes , Victor Guallar , James R. Kincaid , Anabella Ivancich","doi":"10.1016/j.jinorgbio.2025.113199","DOIUrl":"10.1016/j.jinorgbio.2025.113199","url":null,"abstract":"<div><div>We have fully characterized the high-valent intermediates formed during the catalytic cycle of lactoperoxidase (LPO), a heme-containing enzyme present in milk, saliva, tears and airways, playing an important role in mammalian antimicrobial defense. Our combined approach of multifrequency (9–285 GHz) Electron Paramagnetic Resonance (EPR) and resonance Raman (rR) spectroscopies allowed the identification of Tyr21 and Tyr193 as radical sites, having a concerted role with the heme iron and resulting in two distinct [Fe(IV) = O Tyr<sup>•</sup>] intermediates in LPO's catalytic cycle. The presence and strength of H-bonds to Tyr<sup>•</sup> assessed by the C − O stretching mode (v<sub>7a</sub>) and the gx component of the g-tensor detected in their rR and 285-GHz EPR spectra, respectively, induced a difference in pH-dependent stabilization of the [Fe(IV) = O Tyr<sub>21</sub><sup>•</sup>] and [Fe(IV) = O Tyr<sub>193</sub><sup>•</sup>] intermediates. The later resulted from single-step electron tunneling subsequent to the formation of the [Fe(IV) = O Por<sup>•+</sup>] species. A Trp radical contributing to the LPO 285-GHz EPR spectra, discern by the advantageous resolution of g-values, is proposed to facilitate the electron transfer to Tyr193. Our computational analysis (PELE) crucially identified a unique surface binding site for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), close to Tyr21 (shown to host the spin by QM/MM simulations), thus consistent with LPO Tyr<sup>•</sup> pH 5.6 detected in the rR and 285-GHz EPR spectra and the substrate oxidation previously shown by our UV–Vis stopped flow electronic absorption studies. The elucidation of the catalytic electron transfer pathways and the protein-based oxidizing sites is relevant to harnessing the concerted reactivity of heme and specific Tyr/Trp sites in natural biocatalysts.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"277 ","pages":"Article 113199"},"PeriodicalIF":3.2,"publicationDate":"2025-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145839079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-16DOI: 10.1016/j.jinorgbio.2025.113189
Nicholas A. Bainbridge , Leonard G. Luyt
The alpha-emitting isotope actinium-225 is a promising radiometal for developing targeted alpha therapeutics (TAT) to treat malignant diseases. Actinium-225 has a half-life of 9.92 days and a decay chain emitting four alpha-particles. Alpha particle therapeutics are particularly attractive for their precise treatment while sparing surrounding tissue. Effective application of actinium-225 in radiopharmaceutical design requires a chelator that stably coordinates the metal and enables bioconjugation to targeting vectors. BzMacropa, a benzyl-fused diaza-18-crown-6 macrocyclic ligand, exhibits favorable binding properties but is incompatible with peptide-based radiopharmaceuticals synthesized using solid-phase peptide synthesis (SPPS) due to undesirable side reactions such as dimerization, offering limited conjugation flexibility. We report a synthetic route to a protected analogue of BzMacropa integrated with protected tert-butyl ester picolinic acid pendant arms with a free carboxylic acid handle enabling flexible conjugation strategies. The protected chelator was successfully synthesized at the multigram scale and was coupled to the C-X-C chemokine type 4 (CXCR4)-targeting peptide FC122 under SPPS conditions. The resulting conjugate was complexed with lanthanum (III), a non-radioactive surrogate for actinium-225. This work demonstrates a high-yielding, scalable synthesis and a modular platform for developing BzMacropa-peptide conjugates, providing an effective approach to the synthesis of next-generation peptide-based TAT radiopharmaceuticals.
{"title":"Synthesis and lanthanum coordination of a BzMacropa analogue for convenient solid-phase peptide synthesis towards targeted alpha therapeutic discovery","authors":"Nicholas A. Bainbridge , Leonard G. Luyt","doi":"10.1016/j.jinorgbio.2025.113189","DOIUrl":"10.1016/j.jinorgbio.2025.113189","url":null,"abstract":"<div><div>The alpha-emitting isotope actinium-225 is a promising radiometal for developing targeted alpha therapeutics (TAT) to treat malignant diseases. Actinium-225 has a half-life of 9.92 days and a decay chain emitting four alpha-particles. Alpha particle therapeutics are particularly attractive for their precise treatment while sparing surrounding tissue. Effective application of actinium-225 in radiopharmaceutical design requires a chelator that stably coordinates the metal and enables bioconjugation to targeting vectors. BzMacropa, a benzyl-fused diaza-18-crown-6 macrocyclic ligand, exhibits favorable binding properties but is incompatible with peptide-based radiopharmaceuticals synthesized using solid-phase peptide synthesis (SPPS) due to undesirable side reactions such as dimerization, offering limited conjugation flexibility. We report a synthetic route to a protected analogue of BzMacropa integrated with protected tert-butyl ester picolinic acid pendant arms with a free carboxylic acid handle enabling flexible conjugation strategies. The protected chelator was successfully synthesized at the multigram scale and was coupled to the C-X-C chemokine type 4 (CXCR4)-targeting peptide FC122 under SPPS conditions. The resulting conjugate was complexed with lanthanum (III), a non-radioactive surrogate for actinium-225. This work demonstrates a high-yielding, scalable synthesis and a modular platform for developing BzMacropa-peptide conjugates, providing an effective approach to the synthesis of next-generation peptide-based TAT radiopharmaceuticals.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"277 ","pages":"Article 113189"},"PeriodicalIF":3.2,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145832084","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-15DOI: 10.1016/j.jinorgbio.2025.113196
Ashraf Komal, Muhammad Azam, Bibi Saman, Riaz Maira, Muhammad Asim Raza Basra
Metal-based coordination complexes of existing drugs, a step towards to natural system, are gaining the attention of scientists due to their boosted pharmaceutical outcomes with minimal side effect on health. The new mononuclear strontium (Sr) complex of mefenamic acid (Mef) and triethylamine (TEA), [Sr(mef)2(TEA)2].4H2O was manufactured and characterized by using Elemental analysis, UV-VIS spectra, FTIR, SEM-EDX,1H and 13C NMR, TGA, and PXRD. Its free radical scavenging and metal chelating potential of the synthesized compound was assessed by five different in vitro antioxidant assays, while in vivo activities evaluated the anti-inflammatory, analgesic and anxiolytic activities. The studied complex contained Sr(II) metal, which is coordinated to two mefenamato ligands through one oxygen atom of each and two TEA through their nitrogen atoms. In the studied complex, Mef acts as monodentate ligand, and TEA serves as auxiliary ligand. The complex exhibited potent antioxidant activities especially lowest IC₅₀ = 5.6 μM for iron-reducing assay, significant anti-inflammatory response (P < 0.001), and strong analgesic effect (P < 0.0001) compared to the free ligand. In-silico ADMET study showed safe drug profiling for Sr-complex than only ligand and biological prediction of biological activities by PASS online webserver further confirmed the better bioactivities ((Pa > Pi) of pharmacological relevance. With further molecular insight, the synthesized complex may serve as promising lead compound for anti-inflammatory drug development.
{"title":"Synthesis, structural characterization, and biological evaluation of a bioactive strontium (II)–mefenamic acid complex: In-vivo and in-silico insights","authors":"Ashraf Komal, Muhammad Azam, Bibi Saman, Riaz Maira, Muhammad Asim Raza Basra","doi":"10.1016/j.jinorgbio.2025.113196","DOIUrl":"10.1016/j.jinorgbio.2025.113196","url":null,"abstract":"<div><div>Metal-based coordination complexes of existing drugs, a step towards to natural system, are gaining the attention of scientists due to their boosted pharmaceutical outcomes with minimal side effect on health. The new mononuclear strontium (Sr) complex of mefenamic acid (Mef) and triethylamine (TEA), [Sr(mef)<sub>2</sub>(TEA)<sub>2</sub>].4H<sub>2</sub>O was manufactured and characterized by using Elemental analysis, UV-VIS spectra, FTIR, SEM-EDX,<sup>1</sup>H and <sup>13</sup>C NMR, TGA, and PXRD. Its free radical scavenging and metal chelating potential of the synthesized compound was assessed by five different <em>in vitro</em> antioxidant assays, while <em>in vivo</em> activities evaluated the anti-inflammatory, analgesic and anxiolytic activities. The studied complex contained Sr(II) metal, which is coordinated to two mefenamato ligands through one oxygen atom of each and two TEA through their nitrogen atoms. In the studied complex, Mef acts as monodentate ligand, and TEA serves as auxiliary ligand. The complex exhibited potent antioxidant activities especially lowest IC₅₀ = 5.6 μM for iron-reducing assay, significant anti-inflammatory response (<em>P</em> < 0.001), and strong analgesic effect (<em>P</em> < 0.0001) compared to the free ligand. <em>In-silico</em> ADMET study showed safe drug profiling for Sr-complex than only ligand and biological prediction of biological activities by PASS online webserver further confirmed the better bioactivities ((Pa > Pi) of pharmacological relevance. With further molecular insight, the synthesized complex may serve as promising lead compound for anti-inflammatory drug development.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"277 ","pages":"Article 113196"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-15DOI: 10.1016/j.jinorgbio.2025.113197
Turgut Keleş , Zekeriya Biyiklioglu , Gökçe Seyhan , Elif Öz , Rengin Reis , Burak Barut
In this study, manganese (III), cobalt (II), copper (II) and zinc (II) phthalocyanines and their quaternized cationic derivatives bearing morpholine groups in the non-peripheral position were producedand characterized by various spectroscopic techniques such as UV–Vis, FT-IR, 1H and 13C NMR (only for Zn(II)), MALDI-TOF mass spectra. The inhibitory potential of new compounds on AChE and BuChE enzymes was investigated and IC50 values were determined. It was found that water-soluble derivatives showed stronger inhibitory properties. The most effective compounds in AChE inhibition were water-soluble zinc (II) phthalocyanine (ZT6Q) (IC50 = 0.85 ± 0.18 μM) and water-soluble manganese (III) phthalocyanine ZT3Q (IC50 = 2.26 ± 0.38 μM), while ZT6Q showed the strongest effect in BuChE inhibition (IC50 = 5.15 ± 0.48 μM). Lineweaver-Burk analyses revealed that these compounds inhibited both enzymes in a mixed manner. Ki and Kii values were also calculated. In cell viability tests against human neuroblastoma (SH-SY5Y), ZT6Q did not show cytotoxic effects up to 10 μM, on the contrary, they increased cell viability. However, ZT3Q exhibited higher cytotoxicity at 48 h exposure. The findings indicate that these compounds can be evaluated as potential therapeutic agents.
{"title":"Potential water-soluble metal phthalocyanine candidates for the treatment of Alzheimer's disease: Synthesis, cholinesterase enzyme inhibition, kinetic analysis and cytotoxicity against human neuroblastoma (SH-SY5Y)","authors":"Turgut Keleş , Zekeriya Biyiklioglu , Gökçe Seyhan , Elif Öz , Rengin Reis , Burak Barut","doi":"10.1016/j.jinorgbio.2025.113197","DOIUrl":"10.1016/j.jinorgbio.2025.113197","url":null,"abstract":"<div><div>In this study, manganese (III), cobalt (II), copper (II) and zinc (II) phthalocyanines and their quaternized cationic derivatives bearing morpholine groups in the non-peripheral position were producedand characterized by various spectroscopic techniques such as UV–Vis, FT-IR, <sup>1</sup>H and <sup>13</sup>C NMR (only for Zn(II)), MALDI-TOF mass spectra. The inhibitory potential of new compounds on AChE and BuChE enzymes was investigated and IC<sub>50</sub> values were determined. It was found that water-soluble derivatives showed stronger inhibitory properties. The most effective compounds in AChE inhibition were water-soluble zinc (II) phthalocyanine (<strong>ZT6Q)</strong> (IC<sub>50</sub> = 0.85 ± 0.18 μM) and water-soluble manganese (III) phthalocyanine <strong>ZT3Q</strong> (IC<sub>50</sub> = 2.26 ± 0.38 μM), while <strong>ZT6Q</strong> showed the strongest effect in BuChE inhibition (IC<sub>50</sub> = 5.15 ± 0.48 μM). Lineweaver-Burk analyses revealed that these compounds inhibited both enzymes in a mixed manner. <em>K</em><sub><em>i</em></sub> and <em>K</em><sub><em>ii</em></sub> values were also calculated. In cell viability tests against human neuroblastoma (SH-SY5Y), <strong>ZT6Q</strong> did not show cytotoxic effects up to 10 μM, on the contrary, they increased cell viability. However, <strong>ZT3Q</strong> exhibited higher cytotoxicity at 48 h exposure. The findings indicate that these compounds can be evaluated as potential therapeutic agents.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"276 ","pages":"Article 113197"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145787258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-15DOI: 10.1016/j.jinorgbio.2025.113192
Guodong Rao , Lizhi Tao , R. David Britt
Copper proteins are essential for many biological processes, mediating electron transfer and redox catalysis in biochemistry and playing key roles in human health. Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool to probe Cu(II) centers in these proteins, but its application can be complicated by spurious signals arising from adventitious Cu(II) binding to buffers or affinity tags. Here, we systematically examined Cu(II) EPR signals from common biological buffers (Tris, HEPES, MOPS) over pH 6.0–8.0, as well as from a His affinity tag. We show that Tris coordinates Cu(II) across this pH range, whereas HEPES binds Cu(II) weakly at near neutral pH and MOPS essentially does not chelate Cu(II) and rather precipitates Cu(II). These buffer-dependent Cu(II) complexes produce distinct EPR signatures that can interfere with spectral interpretation and spin quantifications. Using two case studies—a bacterial multicopper oxidase (MnxEFG) and Drosophila lysyl oxidase (DmLOX), we show how buffer choices and purification tags influence the observed Cu EPR spectra. In MnxEFG, switching from HEPES to Tris eliminated adventitiously bound Cu(II) and revealed the true copper content and sites. In DmLOX, we identified an EPR signature of Cu(II) bound to the protein's His-tag, which could be minimized by appropriate refolding conditions. These findings highlight the importance of controlling solution chemistry during sample preparation and suggest practical strategies to obtain cleaner, more interpretable spectra. Overall, this work provides guidance for minimizing nonspecific Cu(II) binding and improving the reliability of EPR analysis in studies of copper proteins in biological systems.
{"title":"Deciphering Cu(II) EPR in biological buffers: Implications for copper protein studies","authors":"Guodong Rao , Lizhi Tao , R. David Britt","doi":"10.1016/j.jinorgbio.2025.113192","DOIUrl":"10.1016/j.jinorgbio.2025.113192","url":null,"abstract":"<div><div>Copper proteins are essential for many biological processes, mediating electron transfer and redox catalysis in biochemistry and playing key roles in human health. Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool to probe Cu(II) centers in these proteins, but its application can be complicated by spurious signals arising from adventitious Cu(II) binding to buffers or affinity tags. Here, we systematically examined Cu(II) EPR signals from common biological buffers (Tris, HEPES, MOPS) over pH 6.0–8.0, as well as from a His affinity tag. We show that Tris coordinates Cu(II) across this pH range, whereas HEPES binds Cu(II) weakly at near neutral pH and MOPS essentially does not chelate Cu(II) and rather precipitates Cu(II). These buffer-dependent Cu(II) complexes produce distinct EPR signatures that can interfere with spectral interpretation and spin quantifications. Using two case studies—a bacterial multicopper oxidase (MnxEFG) and <em>Drosophila</em> lysyl oxidase (DmLOX), we show how buffer choices and purification tags influence the observed Cu EPR spectra. In MnxEFG, switching from HEPES to Tris eliminated adventitiously bound Cu(II) and revealed the true copper content and sites. In DmLOX, we identified an EPR signature of Cu(II) bound to the protein's His-tag, which could be minimized by appropriate refolding conditions. These findings highlight the importance of controlling solution chemistry during sample preparation and suggest practical strategies to obtain cleaner, more interpretable spectra. Overall, this work provides guidance for minimizing nonspecific Cu(II) binding and improving the reliability of EPR analysis in studies of copper proteins in biological systems.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"276 ","pages":"Article 113192"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145787259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Two methyl substituents of TriMeOBQDMEN (L0, N,N′-bis(5,6,7-trimethoxy-2-quinolylmethyl)-N,N′-dimethylethylenediamine) have been changed to 3-pentyl, cyclopentyl, and cyclohexyl groups to achieve better Cd2+ selectivity on fluorescence enhancement upon metal ion binding. Bulky alkyl substituents result in weak metal binding affinity of the ligand, which decreases the fluorescence intensity in the presence of excess amount of metal salt, even for Cd(ClO4)2 in DMF-H2O (1:1). During the titration, the protons generated in the presence of excess metal perchlorate inhibit the complexation by protonation of the ligand. Addition of chloride ion minimizes the fluorescence drop in the presence of excess cadmium salt via formation of dinuclear bis(μ-chloro) cadmium complex. Intrinsic fluorescent Cd/Zn selectivity of the ligand can be assessed in DMF-HEPES buffer (1:1, 50 mM HEPES, 0.1 M KCl, pH = 7.5). The use of DMF-borate buffer (1:1, 50 mM M boric acid, 50 mM KCl, pH = 10) effectively discriminates Cd2+ from Zn2+ (IZn/ICd = 0.7 % in the presence of 3 equiv. of metal ions) by utilizing the TriMeOBQDPEN (L2, N,N′-bis(5,6,7-trimethoxy-2-quinolylmethyl)-N,N′-di(3-pentyl)-ethylenediamine) with moderate metal binding affinity. Lowering pH also increases the Cd2+ selectivity (IZn/ICd = 9 % in the presence of 5 equiv. of metal ions) of TriMeOBQDCPEN (L3, N,N′-bis(5,6,7-trimethoxy-2-quinolylmethyl)-N,N′-di(cyclopentyl)ethylenediamine) in DMF-glutarate buffer (1:1, 10 mM 3,3-dimethylglutaric acid, 0.1 M NaCl, pH = 6).
TriMeOBQDMEN的两个甲基取代基（L0， N,N ' -双（5,6,7-三甲氧基-2-喹啉甲基）-N,N ' -二甲基乙二胺）被改变为3-戊基、环戊基和环己基，从而在金属离子结合的荧光增强中获得更好的Cd2+选择性。体积较大的烷基取代基导致配体的金属结合亲和力较弱，在过量金属盐存在时，即使DMF-H2O（1:1）中的Cd(ClO4)2也会降低荧光强度。在滴定过程中，过量金属高氯酸盐存在下产生的质子抑制了配体质子化的络合作用。氯离子的加入通过形成双核双（μ-氯）镉络合物使过量镉盐存在时的荧光下降最小化。在DMF-HEPES缓冲液（1:1,50 mM HEPES, 0.1 M KCl, pH = 7.5）中，可以评估配体的内在荧光Cd/Zn选择性。使用dmf -硼酸缓冲液（1:1,50 mM硼酸，50 mM氯化钾，pH = 10），利用具有中等金属结合亲和力的TriMeOBQDPEN （L2， N,N ' -双（5,6,7-三甲氧基-2-喹啉甲基）-N,N ' -二（3-戊基）-乙二胺），有效区分Cd2+和Zn2+（在3等量金属离子存在下，IZn/ICd = 0.7%）。降低pH也提高了TriMeOBQDCPEN （L3， N，N′-双（5,6,7-三甲氧基-2-喹啉甲基）-N，N′-二（环戊基）乙二胺）在dmf -戊二酸缓冲液（1:1,10 mM 3,3-二甲基戊二酸，0.1 M NaCl, pH = 6）中的Cd2+选择性（在5等量金属离子存在下，IZn/ICd = 9%）。
{"title":"Optimization of bulkiness/dynamicity and pH utilizing N,N′-bis(5,6,7-trimethoxy-2-quinolylmethyl)-N,N′-dialkylethylenediamines as a Cd2+-specific fluorescent sensor","authors":"Yuji Mikata , Saori Yasuda , Poonyawee Suwankeeree , Runo Ozaki , Hideo Konno , Takashi Matsuo , Sunao Shoji","doi":"10.1016/j.jinorgbio.2025.113194","DOIUrl":"10.1016/j.jinorgbio.2025.113194","url":null,"abstract":"<div><div>Two methyl substituents of TriMeOBQDMEN (<strong>L0</strong>, <em>N,N′</em>-bis(5,6,7-trimethoxy-2-quinolylmethyl)-<em>N,N′</em>-dimethylethylenediamine) have been changed to 3-pentyl, cyclopentyl, and cyclohexyl groups to achieve better Cd<sup>2+</sup> selectivity on fluorescence enhancement upon metal ion binding. Bulky alkyl substituents result in weak metal binding affinity of the ligand, which decreases the fluorescence intensity in the presence of excess amount of metal salt, even for Cd(ClO<sub>4</sub>)<sub>2</sub> in DMF-H<sub>2</sub>O (1:1). During the titration, the protons generated in the presence of excess metal perchlorate inhibit the complexation by protonation of the ligand. Addition of chloride ion minimizes the fluorescence drop in the presence of excess cadmium salt via formation of dinuclear bis(μ-chloro) cadmium complex. Intrinsic fluorescent Cd/Zn selectivity of the ligand can be assessed in DMF-HEPES buffer (1:1, 50 mM HEPES, 0.1 M KCl, pH = 7.5). The use of DMF-borate buffer (1:1, 50 mM M boric acid, 50 mM KCl, pH = 10) effectively discriminates Cd<sup>2+</sup> from Zn<sup>2+</sup> (<em>I</em><sub>Zn</sub>/<em>I</em><sub>Cd</sub> = 0.7 % in the presence of 3 equiv. of metal ions) by utilizing the TriMeOBQDPEN (<strong>L2</strong>, <em>N,N′</em>-bis(5,6,7-trimethoxy-2-quinolylmethyl)-<em>N,N′</em>-di(3-pentyl)-ethylenediamine) with moderate metal binding affinity. Lowering pH also increases the Cd<sup>2+</sup> selectivity (<em>I</em><sub>Zn</sub>/<em>I</em><sub>Cd</sub> = 9 % in the presence of 5 equiv. of metal ions) of TriMeOBQDCPEN (<strong>L3</strong>, <em>N,N′</em>-bis(5,6,7-trimethoxy-2-quinolylmethyl)-<em>N,N′</em>-di(cyclopentyl)ethylenediamine) in DMF-glutarate buffer (1:1, 10 mM 3,3-dimethylglutaric acid, 0.1 M NaCl, pH = 6).</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"276 ","pages":"Article 113194"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-15DOI: 10.1016/j.jinorgbio.2025.113193
Shams Aaghaz, Liam R. Marshall, Ivan V. Korendovych
Short peptide-based assemblies combined with metal ions are simple yet effective platforms for catalysis. This study investigates the catalytic properties of de novo amyloid forming peptides, how their catalytic properties change with a range of hydrolysis substrates and the relationship between catalysis and structural characteristics. We have shown that even slight modifications in peptide sequence have a drastic impact on catalytic efficiency and substrate selectivity. We have also established kuncat benchmarks for a 4-methylumbelliferyl ester that is increasingly reported as a model substrate in protein design studies. Our studies have yielded catalytic amyloids which display levels of specific activity on par with those reported for the best de novo globular enzymes. This work expands the understanding of catalytic amyloid function and explores design principles for developing minimal peptide scaffolds with tunable esterase-like activity.
{"title":"Structure-activity relationships in catalytic amyloids","authors":"Shams Aaghaz, Liam R. Marshall, Ivan V. Korendovych","doi":"10.1016/j.jinorgbio.2025.113193","DOIUrl":"10.1016/j.jinorgbio.2025.113193","url":null,"abstract":"<div><div>Short peptide-based assemblies combined with metal ions are simple yet effective platforms for catalysis. This study investigates the catalytic properties of <em>de novo</em> amyloid forming peptides, how their catalytic properties change with a range of hydrolysis substrates and the relationship between catalysis and structural characteristics. We have shown that even slight modifications in peptide sequence have a drastic impact on catalytic efficiency and substrate selectivity. We have also established k<sub>uncat</sub> benchmarks for a 4-methylumbelliferyl ester that is increasingly reported as a model substrate in protein design studies. Our studies have yielded catalytic amyloids which display levels of specific activity on par with those reported for the best <em>de novo</em> globular enzymes. This work expands the understanding of catalytic amyloid function and explores design principles for developing minimal peptide scaffolds with tunable esterase-like activity.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"276 ","pages":"Article 113193"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-15DOI: 10.1016/j.jinorgbio.2025.113195
Naedum I. DomNwachukwu , Matthew D. Bailey , Minrui Luo , Jonathan Schiff , Andrew Brotherton , Bennet Phillips-Sorich , Jonathan Rivnay , Thomas J. Meade
C-reactive protein (CRP) is rapidly upregulated during the early stages of inflammation following injury, infection, or illness and binds to damaged cells in affected tissues, making it an attractive target for imaging. Current clinical assays measure only circulating CRP levels and provide no spatial information about the location of inflammation or its structural impact. Direct visualization of CRP in tissues could enable earlier detection, precise localization of pathology, monitoring of treatment response, and identification of subclinical disease. Here, we report the development of the first CRP-targeted magnetic resonance (MR) imaging contrast agent for molecular imaging of inflammation. We synthesized a gadolinium-labeled gold nanoparticle (AuNP) targeted to CRP through a phosphocholine (PC) ligand, termed PC-Gd@AuNP. The resulting particles were highly uniform (∼2 nm) with a tunable Gd:PC surface ratio. Biophysical characterization demonstrated strong binding affinity for CRP (KD = 135 ± 63.96 nM) and specificity in a complex biological fluid model. PC-Gd@AuNPs displayed high longitudinal relaxivity (8.5 mM−1 s−1 per Gd) and cytotoxicity thresholds (LC₅₀) of 1 μM in HeLa cells and 0.284 μM in HepG2 cells. These findings establish PC-Gd@AuNPs as a promising molecular MRI contrast agent with the potential to directly image CRP deposition in inflamed tissues and advance noninvasive detection and monitoring of inflammatory diseases.
{"title":"Targeted gadolinium-labeled gold nanoparticles for MR imaging of C-reactive protein","authors":"Naedum I. DomNwachukwu , Matthew D. Bailey , Minrui Luo , Jonathan Schiff , Andrew Brotherton , Bennet Phillips-Sorich , Jonathan Rivnay , Thomas J. Meade","doi":"10.1016/j.jinorgbio.2025.113195","DOIUrl":"10.1016/j.jinorgbio.2025.113195","url":null,"abstract":"<div><div>C-reactive protein (CRP) is rapidly upregulated during the early stages of inflammation following injury, infection, or illness and binds to damaged cells in affected tissues, making it an attractive target for imaging. Current clinical assays measure only circulating CRP levels and provide no spatial information about the location of inflammation or its structural impact. Direct visualization of CRP in tissues could enable earlier detection, precise localization of pathology, monitoring of treatment response, and identification of subclinical disease. Here, we report the development of the first CRP-targeted magnetic resonance (MR) imaging contrast agent for molecular imaging of inflammation. We synthesized a gadolinium-labeled gold nanoparticle (AuNP) targeted to CRP through a phosphocholine (PC) ligand, termed PC-Gd@AuNP. The resulting particles were highly uniform (∼2 nm) with a tunable Gd:PC surface ratio. Biophysical characterization demonstrated strong binding affinity for CRP (K<sub>D</sub> = 135 ± 63.96 nM) and specificity in a complex biological fluid model. PC-Gd@AuNPs displayed high longitudinal relaxivity (8.5 mM<sup>−1</sup> s<sup>−1</sup> per Gd) and cytotoxicity thresholds (LC₅₀) of 1 μM in HeLa cells and 0.284 μM in HepG2 cells. These findings establish PC-Gd@AuNPs as a promising molecular MRI contrast agent with the potential to directly image CRP deposition in inflamed tissues and advance noninvasive detection and monitoring of inflammatory diseases.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"276 ","pages":"Article 113195"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145809110","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-15DOI: 10.1016/j.jinorgbio.2025.113190
Alisher Talgatov , Ge Shi , Gurleen Kaur , Xinyang Sun , Kalara D. Gamage , Habiba Afrin , Joshua Rahmon , Elamparuthi Ramasamy , Houston D. Cole , Colin G. Cameron , Wenfang Sun , Sherri A. McFarland
To elucidate how ligand conjugation modulates photophysical behavior and photobiological activity, a new family of bis-terpyridyl Ru(II) complexes bearing oligothienyl-substituted ligands, [Ru(tpy)(tpy-nT)]2+ (n = 0–4; Ru-nT), were synthesized and comprehensively characterized. Progressive thiophene extension produced predictable bathochromic shifts and enhanced intensity of intraligand (ILCT) and metal-to-ligand charge-transfer (MLCT) absorption bands, accompanied by red-shifted phosphorescence (673–818 nm), microsecond excited-state lifetimes, and singlet‑oxygen generation (ΦΔ = 0.46–0.86). The transition from Ru-1T to Ru-2T marked a sharp increase in triplet-state lifetime and ΦΔ, signifying a shift from predominantly MLCT to mixed MLCT/ILCT character. Photocytotoxicity toward SK-MEL-28 melanoma cells revealed wavelength- and oxygen-dependent structure–activity relationships: Ru-2T exhibited exceptional potency under normoxia (EC50 ≈ 0.5 nM, PI >103), whereas Ru-4T retained sub-micromolar activity and high phototoxic indices under hypoxia and red-light irradiation. Increasing thiophene length also enhanced lipophilicity (log Do/w ≈ −1.8 → +0.2), correlating with diminished photocytotoxicity but sustained activity under oxygen limitation. Extension of these studies to microbial models revealed parallel trends – Ru-2T was most effective against Gram-positive E. faecalis V587 (EC50 = 0.17 μM, PI = 216), while Ru-4T showed selective photoactivity toward Gram-negative E. coli W3110 (EC50 = 6.5 μM, PI ≈ 21). Collectively, these results demonstrate that systematic control of conjugation and polarity in Ru(II) bis-terpyridyl frameworks enables tuning of excited-state character, wavelength responsiveness, and biological activity, establishing design principles for oxygen-adaptive photoactive metallodrugs.
{"title":"Conjugation-driven modulation of excited-state dynamics and photobiological activity in Ru(II) bis-terpyridyl oligothienyl complexes","authors":"Alisher Talgatov , Ge Shi , Gurleen Kaur , Xinyang Sun , Kalara D. Gamage , Habiba Afrin , Joshua Rahmon , Elamparuthi Ramasamy , Houston D. Cole , Colin G. Cameron , Wenfang Sun , Sherri A. McFarland","doi":"10.1016/j.jinorgbio.2025.113190","DOIUrl":"10.1016/j.jinorgbio.2025.113190","url":null,"abstract":"<div><div>To elucidate how ligand conjugation modulates photophysical behavior and photobiological activity, a new family of bis-terpyridyl Ru(II) complexes bearing oligothienyl-substituted ligands, <strong>[Ru(tpy)(tpy-<em>n</em>T)]</strong><sup><strong>2+</strong></sup> (<em>n</em> = 0–4; <strong>Ru-</strong><em><strong>n</strong></em><strong>T</strong>), were synthesized and comprehensively characterized. Progressive thiophene extension produced predictable bathochromic shifts and enhanced intensity of intraligand (ILCT) and metal-to-ligand charge-transfer (MLCT) absorption bands, accompanied by red-shifted phosphorescence (673–818 nm), microsecond excited-state lifetimes, and singlet‑oxygen generation (Φ<sub>Δ</sub> = 0.46–0.86). The transition from <strong>Ru-1T</strong> to <strong>Ru-2T</strong> marked a sharp increase in triplet-state lifetime and Φ<sub>Δ</sub>, signifying a shift from predominantly MLCT to mixed MLCT/ILCT character. Photocytotoxicity toward SK-MEL-28 melanoma cells revealed wavelength- and oxygen-dependent structure–activity relationships: <strong>Ru-2T</strong> exhibited exceptional potency under normoxia (EC<sub>50</sub> ≈ 0.5 nM, PI >10<sup>3</sup>), whereas <strong>Ru-4T</strong> retained sub-micromolar activity and high phototoxic indices under hypoxia and red-light irradiation. Increasing thiophene length also enhanced lipophilicity (log <em>D</em><sub>o/w</sub> ≈ −1.8 → +0.2), correlating with diminished photocytotoxicity but sustained activity under oxygen limitation. Extension of these studies to microbial models revealed parallel trends – <strong>Ru-2T</strong> was most effective against Gram-positive <em>E. faecalis</em> V587 (EC<sub>50</sub> = 0.17 μM, PI = 216), while <strong>Ru-4T</strong> showed selective photoactivity toward Gram-negative <em>E. coli</em> W3110 (EC<sub>50</sub> = 6.5 μM, PI ≈ 21). Collectively, these results demonstrate that systematic control of conjugation and polarity in Ru(II) bis-terpyridyl frameworks enables tuning of excited-state character, wavelength responsiveness, and biological activity, establishing design principles for oxygen-adaptive photoactive metallodrugs.</div></div>","PeriodicalId":364,"journal":{"name":"Journal of Inorganic Biochemistry","volume":"277 ","pages":"Article 113190"},"PeriodicalIF":3.2,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145909785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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