Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein.

Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater MnII affinity than FeII affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.

The good, the neutral, and the positive: buffer identity impacts CO2 reduction activity by nickel(ii) cyclam.

Development of new synthetic catalysts for CO2 reduction has been a central focus of chemical research efforts towards mitigating rising global carbon dioxide levels. In parallel with generating new molecular systems, characterization and benchmarking of these compounds across well-defined catalytic conditions are essential. Nickel(ii) cyclam is known to be an active catalyst for CO2 reduction to CO. The degree of selectivity and activity has been found to differ widely across electrodes used and upon modification of the ligand environment, though without a molecular-level understanding of this variation. Moreover, while proton transfer is key for catalytic activity, the effects of varying the nature of the proton donor remain unclear. In this work, a systematic investigation of the electrochemical and light-driven catalytic behaviour of nickel(ii) cyclam under different aqueous reaction conditions has been performed. The activity and selectivity are seen to vary widely depending on the nature of the buffering agent, even at a constant pH, highlighting the importance of proton transfer for catalysis. Buffer binding to the nickel center is negatively correlated with selectivity, and cationic buffers show high levels of selectivity and activity. These results are discussed in the context of molecular design principles for developing increasingly efficient and selective catalysts. Moreover, identifying these key contributors towards activity has implications for understanding the role of the conserved secondary coordination environments in naturally occurring CO2-reducing enzymes, including carbon monoxide dehydrogenase and formate dehydrogenase.

Reasons for Late-Night Eating and Willingness to Change:A Qualitative Study in Pregnant Black Women.

OBJECTIVE: Late-night eating during pregnancy is associated with greater risk for gestational diabetes. The purposes of this study were to describe reasons why women engage in late-night eating and to understand perceptions about changing this behavior. DESIGN: Focus groups using a semi-structured interview script. SETTING: Urban university-affiliated obstetric clinic. PARTICIPANTS: Low-income black women (n = 18) with overweight/obesity at entry to prenatal care. PHENOMENON OF INTEREST: Late-night eating. ANALYSIS: Exhaustive approach coding responses to specific questions. RESULTS: Individual and interpersonal contributors to late-night eating included hunger, altered sleep patterns, fetal movement, and the influence of others. Food choices were largely driven by taste and convenience. Some women reported that they could alter nightly eating patterns, whereas others would consider changing only if late-night eating were associated with a severe illness or disability for the child. CONCLUSIONS AND IMPLICATIONS: There was considerable heterogeneity among the participants of this study regarding reasons for late-night eating during pregnancy and attitudes toward changing this behavior. Although the themes identified from this study cannot be generalized, they may be useful to inform future studies. Future research might develop strategies to overcome individual and social factors that contribute to late-night eating during pregnancy.

A photoactive semisynthetic metalloenzyme exhibits complete selectivity for CO2 reduction in water.

A series of artificial metalloenzymes containing a ruthenium chromophore and [NiII(cyclam)]2+, both incorporated site-selectively, have been constructed within an azurin protein scaffold. These light-driven, semisynthetic enzymes do not evolve hydrogen, thus displaying complete selectivity for CO2 reduction to CO. Electrostatic effects rather than direct excited-state electron transfer dominate the ruthenium photophysics, suggesting that intramolecular electron transfer from photogenerated RuI to [NiII(cyclam)]2+ represents the first step in catalysis. Stern-Volmer analyses rationalize the observation that ascorbate is the only sacrificial electron donor that supports turnover. Collectively, these results highlight the important interplay of elements that must be considered when developing and characterizing molecular catalysts.

Light-Driven Hydrogen Evolution by Nickel-Substituted Rubredoxin.

An enzymatic system for light-driven hydrogen generation has been developed through covalent attachment of a ruthenium chromophore to nickel-substituted rubredoxin (NiRd). The photoinduced activity of the hybrid enzyme is significantly greater than that of a two-component system and is strongly dependent on the position of the ruthenium phototrigger relative to the active site, indicating a role for intramolecular electron transfer in catalysis. Steady-state and time-resolved emission spectra reveal a pathway for rapid, direct quenching of the ruthenium excited state by nickel, but low overall turnover numbers suggest initial electron transfer is not the rate-limiting step. This approach is ideally suited for detailed mechanistic investigations of catalysis by NiRd and other molecular systems, with implications for generation of solar fuels.

Multielectron Chemistry within a Model Nickel Metalloprotein: Mechanistic Implications for Acetyl-CoA Synthase.

The acetyl coenzyme A synthase (ACS) enzyme plays a central role in the metabolism of anaerobic bacteria and archaea, catalyzing the reversible synthesis of acetyl-CoA from CO and a methyl group through a series of nickel-based organometallic intermediates. Owing to the extreme complexity of the native enzyme systems, the mechanism by which this catalysis occurs remains poorly understood. In this work, we have developed a protein-based model for the NiP center of acetyl coenzyme A synthase using a nickel-substituted azurin protein (NiAz). NiAz is the first model nickel protein system capable of accessing three (NiI/NiII/NiIII) distinct oxidation states within a physiological potential range in aqueous solution, a critical feature for achieving organometallic ACS activity, and binds CO and -CH3 groups with biologically relevant affinity. Characterization of the NiI-CO species through spectroscopic and computational techniques reveals fundamentally similar features between the model NiAz system and the native ACS enzyme, highlighting the potential for related reactivity in this model protein. This work provides insight into the enzymatic process, with implications toward engineering biological catalysts for organometallic processes.

An internal electron reservoir enhances catalytic CO2 reduction by a semisynthetic enzyme.

The development of an artificial metalloenzyme for CO2 reduction is described. The small-molecule catalyst [Ni(II)(cyclam)](2+) has been incorporated within azurin. Selectivity for CO generation over H(+) reduction is enhanced within the protein environment, while the azurin active site metal impacts the electrochemical overpotential and photocatalytic activity. The enhanced catalysis observed for copper azurin suggests an important role for intramolecular electron transfer, analogous to native CO2 reducing enzymes.

A narrative review of the associations between six bioactive components in breast milk and infant adiposity.

OBJECTIVE: This narrative review examines six important non-nutritive substances in breast milk, many of which were thought to have little to no biological significance. The overall objective is to provide background on key bioactive factors in breast milk believed to have an effect on infant outcomes (growth and body composition). METHODS: The evidence for the effects of the following six bioactive compounds in breast milk on infant growth outcomes are reviewed: insulin, leptin, adiponectin, ghrelin, interleukin-6, and tumor necrosis factor-α. RESULTS: The existing literature on the effects of breast milk insulin, ghrelin, interleukin-6, and tumor necrosis factor-α and their associations with infant growth and adiposity is sparse. Of the bioactive compounds reviewed, leptin and adiponectin are the most researched. Data reveal that breast milk adiponectin has negative associations with growth in infancy. CONCLUSIONS: There is a need for innovative, well-designed studies to improve causal inference and advance our understanding in the effects of breast milk and its components on offspring growth and body composition. The recommendations provided, along with careful consideration of both known and unknown factors that affect breast milk composition, will help improve, standardize, and ultimately advance this emergent field.

Relatively Low ß-Cell Responsiveness Contributes to the Association of BMI with Circulating Glucose Concentrations Measured under Free-Living Conditions among Pregnant African American Women.

BACKGROUND: Body mass index (BMI, in kg/m(2)) is positively associated with plasma glucose in late pregnancy and with risk of adverse obstetric outcomes. Much of the existing research uses single-clinic measures of plasma glucose, which may not accurately reflect circulating glucose under free-living conditions. Furthermore, little is known about circulating glucose concentrations of African American women, who tend to have poorer diet quality and a greater risk of obstetric complications. OBJECTIVE: The objective of the study was to test the hypothesis that the positive association of BMI in early pregnancy with third-trimester circulating glucose concentrations measured under free-living conditions among African American women would be at least partially attributable to lower ß-cell insulin secretion relative to insulin sensitivity [i.e., lower disposition index (DI)]. METHODS: Using a prospective, observational design, 40 pregnant African American women (mean ± SD age: 23.1 ± 4.0 y; mean ± SD BMI: 28.4 ± 7.5) wore continuous glucose monitors and accelerometers for 3 d at 32-35 wk of gestation and concurrently maintained a food diary to report their self-selected meals. The DI was derived from a 75-g oral glucose tolerance test. Linear regression modeling was used to calculate the association of BMI with the 24-h glucose (GLUC24h) and 2-h (GLUC2hPP) postprandial glucose areas under the curve and with the percentage of time the glucose concentrations were >120 mg/dL. RESULTS: The positive associations between BMI and GLUC24h (standardized ß = 0.36, P = 0.03) and the percentage of time glucose concentrations were >120 mg/dL (standardized ß = 0.40, P = 0.02) were independent of total carbohydrate intake and physical activity and were attenuated when DI was added to the model. The positive association of BMI with GLUC2hPP was attenuated when DI was added to the model, and DI itself was independently associated with GLUC2hPP after self-selected breakfast and dinner (standardized ß = -0.33 and -0.42, respectively; P = 0.01). CONCLUSIONS: The association of BMI with high circulating glucose in free-living pregnant African American women is at least partially attributable to lower ß-cell responsiveness.

Association of late-night carbohydrate intake with glucose tolerance among pregnant African American women.

Obesity and late-night food consumption are associated with impaired glucose tolerance. Late-night carbohydrate consumption may be particularly detrimental during late pregnancy because insulin sensitivity declines as pregnancy progresses. Further, women who were obese (Ob) prior to pregnancy have lower insulin sensitivity than do women of normal weight (NW). The aim of this study is to test the hypothesis that night-time carbohydrate consumption is associated with poorer glucose tolerance in late pregnancy and that this association would be exacerbated among Ob women. Forty non-diabetic African American women were recruited based upon early pregnancy body mass index (NW, <25 kg m(-2) ; Ob, ≥30 kg m(-2) ). Third trimester free-living dietary intake was assessed by food diary, and indices of glucose tolerance and insulin action were assessed during a 75-g oral glucose tolerance test. Women in the Ob group reported greater average 24-h energy intake (3055 kcal vs. 2415 kcal, P < 0.05). Across the whole cohort, night-time, but not day-time, carbohydrate intake was positively associated with glucose concentrations after the glucose load and inversely associated with early phase insulin secretion (P < 0.05). Multiple linear regression modelling within each weight group showed that the associations among late-night carbohydrate intake, glucose concentrations and insulin secretion were present only in the Ob group. This is the first study to report an association of night-time carbohydrate intake specifically on glucose tolerance and insulin action during pregnancy. If replicated, these results suggest that late-night carbohydrate intake may be a potential target for intervention to improve metabolic health of Ob women in late pregnancy.