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At least 559 records · Page 31

TRISO-form HALEU-fueled Experiment for Transport Applications (THETA) Serpent 2 Benchmarking

A number of new advanced reactor designs use TRI-structural ISO-topic (TRISO) fuel. The uranium enrichment for the TRISO fuel is in the 5- to 19.75-weight percent range, which is considered High-Assay Low Enriched Uranium (HALEU). The Nuclear Regulatory Commission and vendors both have a strong interest in benchmarking these advanced reactor designs and having confidence in the predictive capabilities of the computer codes used to model them. In the recent years, Serpent 2 gained several capabilities focused on the reactor applications making it attractive for many reactor vendors.

Critical Experiment↗

TRACE Input Modernization

This work presents a Tom’s Obvious Minimal Language (TOML)-based representation of input for the US Nuclear Regulatory Commission’s TRAC/RELAP Advanced Computational Engine (TRACE) thermal hydraulics code. Implemented using the Workbench Analysis Sequence Processor (WASP), the approach maps traditional TRACE input structures to a hierarchical format composed of named parameters, typed values, and native data collections. The resulting representation preserves TRACE’s existing modeling capabilities while providing a modern, structured interface for model development and management. WASP further extends TOML through a file import directive that supports modular model composition and reusable input organization. In addition, WASP provides extended array data entry convenience with various data repeat and interpolation capabilities. Examples of the new TOML syntax are provided for major TRACE input categories, including hydraulic components, heat structures, control systems, and trip logic. The TOML representation establishes a foundation for improved validation, tooling, automation, and model maintainability while remaining compatible with existing TRACE workflows. To facilitate migration to the TOML-based input format, the TRACE executable now supports conversion of native TRACE input into an intermediate JSON representation. A Python utility subsequently transforms the JSON data into an equivalent TOML model. Lastly, the TRACE executable now supports execution using TOML-formatted input.

Lefebvre, Robert A. [Oak Ridge National Laboratory↗

Fractal landscapes in biological systems: long-range correlations in DNA and interbeat heart intervals

Here we discuss recent advances in applying ideas of fractals and disordered systems to two topics of biological interest, both topics having common the appearance of scale-free phenomena, i.e., correlations that have no characteristic length scale, typically exhibited by physical systems near a critical point and dynamical systems far from equilibrium. (i) DNA nucleotide sequences have traditionally been analyzed using models which incorporate the possibility of short-range nucleotide correlations. We found, instead, a remarkably long-range power law correlation. We found such long-range correlations in intron-containing genes and in non-transcribed regulatory DNA sequences as well as intragenomic DNA, but not in cDNA sequences or intron-less genes. We also found that the myosin heavy chain family gene evolution increases the fractal complexity of the DNA landscapes, consistent with the intron-late hypothesis of gene evolution. (ii) The healthy heartbeat is traditionally thought to be regulated according to the classical principle of homeostasis, whereby physiologic systems operate to reduce variability and achieve an equilibrium-like state. We found, however, that under normal conditions, beat-to-beat fluctuations in heart rate display long-range power law correlations.

Non-NASA Center↗

A model to assess Zircaloy’s mechanical property changes following a transient beyond critical heat flux

Maintaining the integrity of nuclear fuel rods is essential for ensuring public health and safety in nuclear power generation. During reactor operation, this integrity is confirmed by demonstrating compliance with established regulatory acceptance criteria. For moderate-frequency events, such as limiting transients and anticipated operational occurrences (AOOs), the current fuel integrity criterion is based on preventing boiling transition. This criterion assumes that prevention of boiling transition will prevent excessive cladding heating and, thus, fuel failure during normal operations. While conservative, this approach places significant constraints on core design, fuel cycle economics, and a plant’s ability to perform major power uprates, leading to suboptimal fuel utilization and inefficient carbon-free energy production. A more efficient approach could be achieved by revising the failure criterion to a material-specific limit rather than strictly preventing the boiling transition, since boiling transition per se is not a cause of fuel cladding failure. Here, as a result, a new licensing framework based on material properties, termed time-at-temperature (t@T), is needed. This approach would allow for brief periods of post–critical heat flux operation during an AOO without compromising safety. Implementing the t@T licensing strategy requires a robust technical foundation in material properties, which must be established through comprehensive data collection on both unirradiated and irradiated fuel and cladding materials. This foundation would enable the development of a safety basis that ensures safe operation while providing greater flexibility and efficiency for reactor operation. This paper documents a thorough review of the available data to establish a baseline knowledge that can inform the development of cladding mechanical models, as well as identify experimental data gaps that need to be addressed in future research. Machine learning and data informatics were utilized to extract the importance of parameters on the t@T parameter. Industry tools were used to perform baseline analyses to define the relevant transient conditions for data analysis. The subsequent review successfully identified applicable experimental data, as well as sufficient data to evaluate changes in cladding mechanical properties following an AOO transient. Rather than developing new models, this work coupled existing irradiation annealing and recrystallization models to calculate changes in hardness, yield stress, and ultimate tensile stress following an AOO event. The findings from this review were summarized to highlight the experimental data needs required to fill remaining gaps and support the development of future t@T licensing methodologies.

Cladding performance↗

Projected increases in wildfires may challenge regulatory curtailment of PM 2.5 over the eastern US by 2050

Anthropogenic contribution to the overall fine particulate matter (PM 2.5 ) concentrations has been declining sharply in North America. In contrast, a steep rise in wildfire-induced air pollution events with recent warming is evident in the region. Here, based on coupled fire–climate–ecosystem model simulations, summertime wildfire-induced PM 2.5 concentrations are projected to nearly double in North America by the mid-21st century compared to the present. More strikingly, the projected enhancement in fire-induced PM 2.5 (~1–2 µg m -3 ) and its contribution (~15 %–20 %) to the total PM 2.5 are distinctively significant in the eastern US. This can be attributed to downwind transport of smoke from future enhancement of wildfires in North America to the eastern US and associated positive climatic feedback on PM 2.5 , i.e., perturbations in circulation, atmospheric stability, and precipitation. Therefore, the anticipated reductions in PM 2.5 from regulatory controls on anthropogenic emissions could be significantly compromised in the future in the densely populated eastern US.

54 ENVIRONMENTAL SCIENCES↗

Open Power System Datasets and Open Simulation Engines: A Survey Toward Machine Learning Applications

A major factor behind the success of machine learning (ML) models in multiple domains is the availability and accessibility of large, labeled, and well-organized datasets for training and benchmarking. In comparison, power grid datasets face three major challenges: (i) real-world data is often restricted by regulatory constraints, privacy reasons, or security concerns, making it difficult to obtain and work with; (ii) synthetic datasets, which are created to address these limitations, often have incomplete information and are released using specialized tools, making them inaccessible to the broader community; and, (iii) input-output datasets are difficult to generate through simulation for non-experts because open-source simulators are not known outside the power system community. This survey addresses these challenges by serving as an entry point to publicly available datasets and simulators for researchers venturing in this area. We review the current landscape of open-source power network data, machine models, consumer demand profiles, renewable generation data, and inverter models. We also examine open-source power system simulators, which are crucial for generating high-quality, high-fidelity power grid datasets. We aim to provide a foundation for overcoming data scarcity and advance towards a structured web of datasets and simulators to support the development of ML for power systems.

42 ENGINEERING↗

BISON Validation to In-Situ Cladding Burst Test and High Burnup LOCA Experiments

The process to develop and qualify nuclear fuels for commercial nuclear application requires fundamental material development, characterization, and design; out-of-pile testing on unirradiated materials; integral fuel rod irradiations, testing, and post irradiation examinations (PIEs); and transient analyses. The historical approach depends on the generation of large empirical datasets and series of integral fuel rod irradiations, and this approach ultimately takes approximately 20 years—or sometimes longer—to acquire data through extensive sequential testing. Thus, the qualification and eventual deployment of new fuel systems constitute a long and drawn-out process. However, recent technological advancements have provided researchers the opportunity to perform out-of-cell, in-situ measurements to assess material performance for the duration of the experiment. One such example of this capability is the use of digital image coordination and thermal imaging to assess Zircaloy cladding performance under a simulated loss-of-coolant accident (LOCA) transient condition. In general, the in-situ measurements provide high-fidelity strain, strain rates, and temperature surface maps. This is critical for the United States nuclear industry, which is actively developing a technical basis to support extending the peak rod average burnup from 62 to ~75 GWd/tU and the deployment of accident-tolerant fuel. However, the Nuclear Regulatory Commission, through their research information letter, outlined a number of technical issues for the industry to address before extending burnup. One topic of specific interest is understanding the cladding balloon and rupture geometry during the LOCA heatup phase. Leveraging these advanced in-situ capabilities, this work intends to use insitu data generated from a simulated LOCA in the Severe Accident Test Station at Oak Ridge National Laboratory to better understand high-temperature creep and its impact on Zircaloy balloon and rupture performance. This work used the BISON fuel performance code to compare BISON’s high-temperature creep model predictions to in-situ data and identify limitations and gaps within the model. Additionally, BISON will subsequently be used to simulate relevant high-burnup LOCA tests. The results will be analyzed and compared to the available post-test data in a manner consistent with the approach outlined in the Nuclear Regulatory Commission research information letter.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Magnetic Microspheres and Tissue Model Studies for Therapeutical Applications

Hyperthermia is a well known cancer therapy and consists of heating a tumor region to the elevated temperatures in the range of 40-45 C for an extended period of time (2-8 hours). This leads to thermal inactivation of cell regulatory and growth processes with resulting widespread necrosis, carbonization and coagulation. Moreover, heat boosts the tumor response to other treatments such as radiation, chemotherapy or immunotherapy. Of particular importance is careful control of generated heat in the treated region and keeping it localized. Higher heating, to about 56 C can lead to tissue thermo-ablation. With accurate temperature control, hyperthermia has the advantage of having minimal side effects. Several heating techniques are utilized for this purpose, such as whole body hyperthermia, radio-frequency (RF) hyperthermia, ultrasound technique, inductive microwave antenna hyperthermia, inductive needles (thermoseeds), and magnetic fluid hyperthermia (MFH).MFH offers many advantages as targeting capability by applying magnets. However, this technology still suffers significant inefficiencies due to lack of thermal control. This paper will provide a review of the topic and outline the ongoing work in this area. The main emphasis is in devising ways to overcome the technical difficulty in hyperthermia breast therapy of achieving a uniform therapeutic temperature over the required region of the body and holding it steady for an extended period (2-3 hours). The basic shortcomings of the presently utilized heating methods stem from the non-uniform thermal properties of the tissue and the point heating characteristics of the techniques without any thermal control. Our approach is to develop a novel class of magnetic fluids, which have inherent thermoregulating properties. We have identified a few magnetic alloys which can serve as suitable nano to micron-size particle material. The objective is to synthesize, characterize and evaluate the efficacy of Thermo Regulating Magnetic Fluids (TRMF) for hyperthermia therapy. The development of a tissue model and testing the fluid dynamics of particle motion, settling, distribution in the tissue matrix and heat generation will be discussed.

Ramachandran, N.↗

Rubisco regulation in response to altered carbon status in the cyanobacterium Synechococcus elongatus PCC 7942

Abstract Photosynthetic organisms possess a variety of mechanisms to achieve balance between absorbed light (source) and the capacity to metabolically utilize or dissipate this energy (sink). While regulatory processes that detect changes in metabolic status/balance are relatively well studied in plants, analogous pathways remain poorly characterized in photosynthetic microbes. Here, we explored systemic changes that result from alterations in carbon availability in the model cyanobacterium Synechococcus elongatus PCC 7942 by taking advantage of an engineered strain where influx/efflux of a central carbon metabolite, sucrose, can be regulated experimentally. We observed that induction of a high-flux sucrose export pathway leads to depletion of internal carbon storage pools (glycogen) and concurrent increases in estimates of photosynthetic activity. Further, a proteome-wide analysis and fluorescence reporter-based analysis revealed that upregulated factors following the activation of the metabolic sink are concentrated on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and auxiliary modules involved in Rubisco maturation. Carboxysome number and Rubisco activity also increased following engagement of sucrose secretion. Conversely, reversing the flux of sucrose by feeding exogenous sucrose through the heterologous transporter resulted in increased glycogen pools, decreased Rubisco abundance, and carboxysome reorganization. Our data suggest that Rubisco activity and organization are key variables connected to regulatory pathways involved in metabolic balancing in cyanobacteria.

Plant Sciences↗

MACCS (MELCOR Accident Consequence Code System) User Guide

The MELCOR Accident Consequence Code System (MACCS) is used by Nuclear Regulatory Commission (NRC) and various national and international organizations for probabilistic consequence analysis of nuclear power accidents. This User Guide is intended to assist analysts in understanding the MACCS/WinMACCS model and to provide information regarding the code. This user guide version describes MACCS Version 3.10.0. Features that have been added to MACCS in subsequent versions are described in separate documentation. This User Guide provides a brief description of the model history, explains how to set up and execute a problem, and informs the user of the definition of various input parameters and any constraints placed on those parameters. This report is part of a series of reports documenting MACCS. Other reports include the MACCS Theory Manual, MACCS Verification Report, Technical Bases for Consequence Analyses Using MACCS, as well as documentation for preprocessor codes including SecPop, MelMACCS, and COMIDA2. PAPERWORK REDUCTION ACT STATEMENT The NUREG does not contain information collection requirements and, therefore, is not subject to the requirements of the Paperwork Reduction Act of 1995 (44 USC 3501, et seq.). PUBLIC PROTECTION NOTIFICATION The NRC may not conduct or sponsor, and a person is not required to respond to, a request for information or an information collection requirement unless the requesting document displays a currently valid OMB control number. ACKNOWLEDGEMENTS Contributions to this User Guide were received from NRC and Sandia National Laboratories (SNL) project managers, technical experts, and code authors dedicated to the production of a valuable resource for the MACCS user community. Instructions and guidance included herein were developed over many years and include advancements in the code that provide users the ability to develop complex consequence modeling scenarios. WinMACCS and many of the early MACCS developments were due to vision of an earlier Project Manager, Jocelyn Mitchell. Jonathan Barr and AJ Nosek also contributed to the development of this report. The current NRC Project Manager, Salman Haq, provided the leadership to ensure this document was completed. Several other NRC and Sandia staff provided insights supporting development of the MACCS code and of this document.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

MACCS (MELCOR Accident Consequence Code System) User Guide -- Version 4.0

The MELCOR Accident Consequence Code System (MACCS) is used by Nuclear Regulatory Commission (NRC) and various national and international organizations for probabilistic consequence analysis of nuclear power accidents. This User Guide is intended to assist analysts in understanding the MACCS/WinMACCS model and to provide information regarding the code. This user guide version describes MACCS Version 4.0. Features that have been added to MACCS in subsequent versions are described in separate documentation. This User Guide provides a brief description of the model history, explains how to set up and execute a problem, and informs the user of the definition of various input parameters and any constraints placed on those parameters. This report is part of a series of reports documenting MACCS. Other reports include the MACCS Theory Manual, MACCS Verification Report, Technical Bases for Consequence Analyses Using MACCS, as well as documentation for preprocessor codes including SecPop, MelMACCS, and COMIDA2.

97 MATHEMATICS AND COMPUTING↗

MACCS (MELCOR Accident Consequence Code System) User Guide Version 4.0, Revision 1

The MELCOR Accident Consequence Code System (MACCS) is used by Nuclear Regulatory Commission (NRC) and various national and international organizations for probabilistic consequence analysis of nuclear power accidents. This User Guide is intended to assist analysts in understanding the MACCS/WinMACCS model and to provide information regarding the code. This user guide version describes MACCS Version 4.0. Features that have been added to MACCS in subsequent versions are described in separate documentation. This User Guide provides a brief description of the model history, explains how to set up and execute a problem, and informs the user of the definition of various input parameters and any constraints placed on those parameters. This report is part of a series of reports documenting MACCS. Other reports include the MACCS Theory Manual, MACCS Verification Report, Technical Bases for Consequence Analyses Using MACCS, as well as documentation for preprocessor codes including SecPop, MelMACCS, and COMIDA2.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

MACCS User Guide (V.4.2)

MACCS is used by the Nuclear Regulatory Commission (NRC) and various national and international organizations for probabilistic consequence analysis of nuclear power accidents. This User Guide is intended to assist analysts in understanding the MACCS/WinMACCS model and to provide information regarding the code. This user guide version describes MACCS Version 4.2. This User Guide provides a brief description of the model history, explains how to set up and execute a problem, and informs the user of the definition of various input parameters and any constraints placed on those parameters. This report is part of a series of reports documenting MACCS. Other reports include the MACCS Theory Manual, MACCS Verification Report, Technical Bases for Consequence Analyses Using MACCS, as well as documentation for preprocessor codes including SecPop, MelMACCS, and COMIDA2.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

The behavior of renal-regulating hormones during hypogravic stress

The regulation of fluid and electrolyte behavior during space flight is believed to be under control, in large part, of a group of hormones which have their major effects on renal excretion. The hormones studied include renin-angitensin, aldosterone, and antidiuretic hormone (ADH). The regulatory systems of these renal-regulating hormones as they act individually and in concert with each other are analyzed. The analysis is based on simulations of the mathematical model of Guyton. A generalized theory is described which accounts for both short-term and long-term behavior of this set of hormones.

Leonard, J. I.↗

Using Calibrated Water Data for Preliminary Validation of the SRT Code for Advanced Reactors

Recent interest and corresponding progress worldwide regarding advanced nuclear reactors has renewed focus on their performance and related safety assessments. Specifically, the U.S. Nuclear Regulatory Commission has emphasized the importance of mechanistic approaches to source term analysis for advanced reactor licensing applications, which attempt to realistically account radionuclide transport and retention phenomena. Further model development is required due to the numerous and complex physical and chemical phenomena associated with mechanistic source term analyses. Reflecting the need for modeling advancement, Argonne National Laboratory developed a mechanistic source term analysis tool for sodium fast reactors. The Simplified Radionuclide Transport (SRT) code describes fuel pin failure (for simulating the initial condition at the point of fuel pin breach), bubble scrubbing, deposition, leakage and following environmental impact. In the current work, a validation study of the SRT bubble scrubbing model is performed using a water-loop experiment performed at the University of Wisconsin-Madison. Through the analysis, the approach and fundamental bubble scrubbing models in SRT, which examine the removal of aerosols within the bubble as it is transported through a pool, have been widely evaluated. The results of the assessment demonstrate a high level of agreement in the regions of greater aerosol size. In the parameter range of minimal aerosol removal, the simulation slightly underpredicts the experiment results; however, considering the scale of plots and huge uncertainties inherently included, the deviation can be judged to be minor and would produce a conservative result. In addition, uncertainty analysis has been further refined to reflect the experimental distribution of parameters including aerosol sizes, which induces a span of performance for each representative aerosol size. Based upon the initial validation results along with uncertainty effects, SRT is expected to provide meaningful insights for the analysis of bubble scrubbing. Future sodium-loop tests will provide further validation basis.

Kam, Dong Hoon↗

Williston Basin Resource Study for Commercial-Scale Subsurface Hydrogen Storage

The Energy & Environmental Research Center (EERC), in partnership with the U.S. Department of Energy (DOE) National Energy Technology Laboratory (NETL), the EERC’s State Energy Research Center (SERC), MPLX Operations LLC, and TC Energy Development Holdings Inc. (a subsidiary of TC Energy Corporation), studied the potential for subsurface hydrogen storage and recovery in the Williston Basin of western North Dakota. The project’s goal was to evaluate the feasibility of large-scale, secure geologic H 2 storage to support future hydrogen generation, storage, and use. This work included laboratory testing, H 2 –rock–fluid exposure experiments, literature reviews on H 2 embrittlement, and reservoir modeling and simulations. The study included an assessment of storage potential across three types of storage reservoirs using both reservoir simulation and DOE’s web-based tool SHASTA-HELP (Subsurface Hydrogen Assessment, Storage, and Technology Acceleration – Hydrogen Estimator for Logistical Planning), as well as investigation of potential H 2 production and markets for commercial-scale deployment. Building on prior EERC gas storage research, three storage options were selected for detailed evaluation: the Broom Creek Formation (a clastic saline reservoir), the Dickinson Lodgepole Mounds (DLM) complex (carbonate mud mound structures) of the Lodgepole Formation (an active oil and gas producing reservoir), and the Dunham Salt Interval of the Piper Formation (to be used for engineered salt cavern development). These targets were prioritized based on prior EERC research using datasets related to seal capacity, reservoir quality, mechanical integrity, and injectivity. Exposure tests on Broom Creek and DLM samples showed mineral dissolution and precipitation that increased brine salinity and altered reservoir rock surfaces. Although these results provide useful insight, they are limited by small sample sizes and short-term (30-day) exposure, requiring further study to assess long-term storage integrity. Salt formations were not tested because of their known nonreactivity and established mechanical stability. Results of reservoir simulations performed for a single site demonstrated that the Broom Creek Formation may be capable of receiving up to 42,000 tonnes of injected H 2 over 7 months via one well. H 2 recovery took place over 5 months, resulting in approximately 26,000 tonnes (~62% without cushion gas [CG]). This work suggests water production may be important and subsequent cycles of injection and production may perform more efficiently; however, significant site-specific work in the future is needed to assess actual reservoir performance of injection and withdrawal of H 2 storage. For oil reservoir potential, a multiple-well model was used to simulate injection of approximately 32,000 tonnes of H 2 into a single wellbore while simultaneously producing in place reservoir fluids from four offset wells to maintain reservoir pressure. The simulation results suggested a high recovery (~98%); in addition, cost advantages through existing infrastructure could be realized. Challenges in this reservoir include vi managing gas purity and leakage risks. In both scenarios, production of H 2 takes place in a single-well scenario with 10 cycles (7 months of injection and 5 months of production) over 10 years. Finally, the use of engineered caverns in the Dunhan Salt was evaluated, and the results suggest that while they have a smaller capacity (<1000 tonnes per cavern), they exhibit nearly complete gas recovery (>99%), fast response times, and low purity risk. While caverns in North Dakota may be smaller in capacity, fields can be developed in galleries to accommodate the volumetric needs and rapid turnaround times necessary to meet market demands. Geographic limitations and thin salt intervals in North Dakota may represent less total storage potential than salt domes elsewhere, but significant opportunities exist to expand this market for gas storage in North Dakota. A basinwide assessment was performed to estimate a first-of-its-kind value for H 2 storage on a large scale. DOE’s SHASTA-HELP, combined with EERC simulation work, was used to perform the assessment. Estimated H 2 storage potential varied widely for each formation type. The Broom Creek saline formation was estimated to have a storage potential of approximately 1.7–90.5 million tonnes (MMt). The DLM oil reservoirs were estimated to have 0.07–0.19 MMt of capacity. Notably, each of these estimates relies on significant assumptions regarding reservoir thickness, porosity, permeability, and CG needed for operation. Much research is needed to understand the true site-specific storage resource potential of each formation. Using the Dunham Salt Interval for cavern development may result in as much as 4.79 MMt (up to 2.87 MMt working gas) of H 2 storage potential. An important note for consideration is that the values presented here need significantly more geological characterization and engineering assessments prior to gaining confidence in performance. This will be a focal point for future research and development needs. The basinwide evaluation also indicated that North Dakota has significant H 2 generation potential, with estimates up to a possible 13 MMt annually, suggesting a substantial opportunity for H 2 market development and thus the need for commercial-scale H 2 storage to facilitate growth and resilience. Key Recommendations 1. Conduct detailed site characterization (3D seismic, well logs, core sampling) to reduce geologic uncertainty. 2. Perform techno-economic analyses incorporating market, regulatory, and incentive frameworks. 3. Investigate long-term interactions among H 2 , CGs, well materials, and formations to assess risks. 4. Develop pilot- and field-scale demonstrations to validate models and establish best practices.

03 NATURAL GAS↗

Two TonB-dependent transporters in Methylosinus trichosporium OB3b are responsible for uptake of different forms of methanobactin and are involved in the canonical ‘copper switch’

Copper is an important component of methanotrophic physiology as it controls the expression and activity of alternative forms of methane monooxygenase (MMO). To collect copper, some methanotrophs secrete a chalkophore or copper-binding compound called methanobactin (MB). MB is a ribosomally synthesized post-translationally modified polypeptide (RiPP) that, after binding copper, is collected by MbnT, a TonB-dependent transporter (TBDT). Structurally different forms of MB have been characterized, and here we show that different forms of MB are collected by specific TBDTs. Further, we report that in the model methanotroph, Methylosinus trichosporium OB3b, expression of the TBDT required for uptake of a different MB made by Methylocystis sp. strain SB2 (MB-SB2), is induced in the presence of MB-SB2, suggesting that methanotrophs have developed specific machinery and regulatory systems to actively take up MB from other methanotrophs for copper collection. Moreover, the canonical “copper-switch” in Ms. trichosporium OB3b that controls expression of alternative MMOs is apparent if one of the two TBDTs required for MB-OB3b and MB-SB2 uptake is knocked out, but is disrupted if both TBDTs are knocked out. Furthermore, these data indicate that MB uptake, including the uptake of exogenous MB, plays an important role in the copper switch in M. trichosporium OB3b and thus overall activity. Based on these data, we propose a revised model for the “copper-switch” in this methanotroph that involves MB uptake.

54 ENVIRONMENTAL SCIENCES↗

Identification of the prooxidant site of human ceruloplasmin: a model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

NASA Discipline Regulatory Physiology↗