Understanding the oxidation states of transition metals in metalloproteins is critical for elucidating their biochemical functions. Among the available spectroscopic techniques, X-ray Absorption Near Edge Structure (XANES) spectroscopy offers a powerful, element-specific method for probing the electronic environment and oxidation state of metal centers in biological macromolecules. The shape, position, and intensity of the absorption edge in a XANES spectrum can provide direct insights into the valence state and coordination geometry of the metal ion, making it a particularly valuable tool for studying redox-active metalloproteins under near-physiological conditions.In this study, we apply XANES spectroscopy to investigate the copper oxidation states in amicyanin, a well- characterized Type-I copper protein, in its crystalline form. Type-I copper sites are highly conserved among various redox proteins found in bacteria, plants, and animals, and are known for their intense blue color due to strong ligand-to-metal charge transfer transitions. These proteins serve as efficient electron transfer (ET) agents, playing vital roles in biological processes such as respiration and metabolism.Amicyanin, a representative member of the cupredoxins family, features a single copper ion coordinated by a distorted tetrahedral geometry with three strong equatorial ligands (a cysteine and two histidines) and one weak axial ligand, typically methionine. It functions as an electron acceptor for the enzyme methylamine dehydrogenase and transfers it to cytochrome C551i.Our objective is to acquire XANES spectra from single crystals of amicyanin in both oxidized and reduced states. These spectra will be analyzed to determine the oxidation state of the copper center and to detect any associated changes in the local electronic environment. Results from XANES will be compared with structure from single-crystal X-ray crystallography, which provides complementary structural information about the copper coordination sphere.This comparative study aims to deepen our understanding of how changes in oxidation state correlate with local structural features at the active site, enhancing our ability to interpret redox mechanisms in copper proteins at atomic resolution. Results from these complementary techniques will be presented, highlighting how their synergy enables a deeper insight into structure-function relationships in metalloproteins.

The workshop titled `X-ray-based technologies in emerging fuel cell research', organized by Vivian Stojanoff from Brookhaven National Laboratory (BNL) and Narayanasami Sukumar from Cornell University/Advanced Photon Source-Northeastern Collaborative Access Team, was a notable segment of the National Synchrotron Light Source II and Center for Functional Nanomaterials Users' Meeting held 13–17 May 2024. This one-day event, on 13 May 2024, at BNL in New York, aimed to bring together researchers, beamline scientists, management and developers to propel fuel cell technology forward using model systems inspired by natural photosynthesis and redox enzymes. This summary encapsulates the key discussions, advancements and future implications of the workshop.

Photosynthesis uses light energy to split water into protons, electrons, and oxygen, ultimately sustaining heterotrophic life. There are several studies on photosynthesis to understand and design artificial photosynthesis where solar energy is converted into solar fuels. Attempts are also being made to create fuel cell by trapping enzymes in the nanoconfined environment (enzyme based fuel cell). However, in both cases, the successful creation of fuel cells is depending on thorough understanding of electron movement between the components and the stability of the electrodes in long term.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic ‘gatekeeper’ enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp–Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg–Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

The Northeastern Collaborative Access Team (NE-CAT) develops and operates advanced synchrotron X-ray beamlines to support high-impact structural biology research. With specialized instrumentation and deep expertise, NE-CAT serves a global user base, facilitating structural determination of challenging macromolecular systems.Following the completion of the Advanced Photon Source (APS) upgrade, NE-CAT has implemented key improvements to ensure optimal performance, flexibility, and reliability in data collection. These include a high- speed storage infrastructure and the deployment of RAPDv2—a next-generation data processing suite that offers seamless integration, scalable performance, and automated workflows for molecular replacement (MR) and single- wavelength anomalous dispersion (SAD) experiments.Ongoing developments include the installation of a new monochromator for beamline 24-ID-C, the integration of MD3 micro-diffractometers for enhanced precision, and the deployment of a custom-built automounter capable of holding 30 sample pucks. Additionally, we are introducing an upgraded remote user interface to enable fully automated data collection and expanding support for room-temperature data acquisition experiments.Currently, NE-CAT operates beamline 24-ID-E, a fixed-energy (12.662 keV) undulator beamline optimized for remote use. It’s equipped with an MD2 micro-diffractometer, ALS-style 14-puck automounters, and the RAPDv2 platform. NE-CAT offers 24/7 user support and secure, flexible data access through Globus or a custom Python sync script. Beamline 24-ID-C is under development and will soon launch with fully redesigned systems and enhanced capabilities.NE-CAT is funded by a P30 grant from the National Institute of General Medical Sciences (NIGMS), with additional support from member institutions. We welcome collaboration and community input as we continue advancing resources to meet the evolving needs of structural biology.

Flavin-dependent halogenases (FDHs) catalyze selective halogenation of electron-rich aromatic compounds without the need for harsh oxidants required by conventional oxidative halogenation reactions. Predictive models for halogenase site selectivity could greatly improve their utility for chemical synthesis. Toward this end, we analyzed the structures and selectivity of three halogenase variants evolved to halogenate tryptamine with orthogonal selectivity. Crystal structures and reversion mutations revealed key residues involved in altering halogenase selectivity. Density functional theory calculations and molecular dynamics simulations are both consistent with hypohalous acid as the active halogenating species in FDH catalysis. This model was used to accurately predict the site selectivity of halogenase variants toward different synthetic substrates, providing a valuable tool for implementing halogenases in biocatalysis efforts.

Electron transfer (ET) through and between proteins is a frequently occurring biological process in both prokaryotes and eukaryotes. Understanding the principle behind the ET will help to understand the important biological processes like respiration and photosynthesis. Several studies have been carried out on amicyanin and variants created by site-directed mutagenesis as a model system to study ET, which revealed several factors affecting this process. Very fine structural features like the position of hydrogen atoms, orientation of water molecules, distribution of valance electrons are essential for understanding the biological activities like ET in detail. An earlier joint x-ray and neutron study on amicyanin reveal location of hydrogen atoms of every residue and orientation of water molecules (with both hydrogens) at oxidized state. The current study applied the unique feature of x-ray crystallography, valance electron density (charge density) to visualize ET through amicyanin. As the current study involve ultra-high-resolution x-ray data, it details the features which can be observed only at this resolution such as location of hydrogen atoms and orientation of water molecules. Data sets were collected for oxidized amicyanin (0.65Å), reduced amicyanin at pH 4.4 (0.67Å) and reduced amicyanin at pH 7.7 (0.75Å). PHENIX, SHELX and MoPro programs were used for refinement and analysis. The analysis of oxidized amicyanin data showed the experimentally determined location of hydrogen atoms for various residues and water molecules. The study analyzed ET hot spots in amicyanin, particularly around the copper site and along the electron transfer path in terms of valence electron density. Residual and static deformation electron density maps were calculated along with Laplacian maps. It revealed deformation of bond density for various important residues. Details will be presented.

Vitamin K antagonists are widely used anticoagulants that target vitamin K epoxide reductases (VKOR), a family of integral membrane enzymes. To elucidate their catalytic cycle and inhibitory mechanism, we report 11 x-ray crystal structures of human VKOR and pufferfish VKOR-like, with substrates and antagonists in different redox states. Substrates entering the active site in a partially oxidized state form cysteine adducts that induce an open-to-closed conformational change, triggering reduction. Binding and catalysis are facilitated by hydrogen-bonding interactions in a hydrophobic pocket. The antagonists bind specifically to the same hydrogen-bonding residues and induce a similar closed conformation. Thus, vitamin K antagonists act through mimicking the key interactions and conformational changes required for the VKOR catalytic cycle.