Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer in photosynthesis and respiration to catalysis in industrial and fuel cell research. ...These functions are accomplished in nature by only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or electron-transfer properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centres or fuel cell catalysts for energy conversion. Here we show that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of azurin is now predictable across the full range of cupredoxin potentials.
Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing ...proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.
A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural ...data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by ~110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ~100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.
In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°′) of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F ...mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from Pseudomonas aeruginosa. These variants were found to differentially influence the E°′ of T1Cu, with M13F Az decreasing E°′, M44F Az increasing E°′, and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases E°′ by 26 mV relative to WT-Az, which is very close to the combined effect of E°′ by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.
Display omitted
•Higher BPA exposure was associated with lower semen quality.•Higher BPS exposure was associated with lower sperm motility.•Higher exposure to BPA, BPF and BPS mixtures were related ...to declined semen quality.
Human studies on association between bisphenol A (BPA) exposure and semen quality, mostly based on single urinary measurement, are inconsistent. There is limited human evidence on BPA analogues such as bisphenol F (BPF) and bisphenol S (BPS), and little is known on potential effects of bisphenol mixtures. We aimed to explore whether individual or mixtures of BPA, BPS and BPF assessed in repeated urinary measurements were associated with semen quality among 984 Chinese men from an infertility clinic. We found that higher BPA exposure was associated with increased odds ratios (ORs) of having below-reference sperm concentration, total sperm count, progressive motility and total motility (all P for trends < 0.05). Higher BPS exposure was associated with increased ORs of having below-reference progressive motility and total motility (both P for trends = 0.02); the ORs comparing extreme quartiles were 1.62 (95% CI: 1.07, 2.43) and 1.57 (95% CI: 1.06, 2.33), respectively. Elevated risks for each outcome were also observed when bisphenol mixtures were at ≥ 55th percentiles. For semen quality parameters modeled as continuous outcomes, inverse associations with individual BPA and BPS and bisphenol mixtures were still estimated. Our results suggested that higher exposure to individual BPA and BPS and bisphenol mixtures were associated with impaired semen quality.
Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been ...fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP), and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation enhanced Mn(II)-binding affinity, as evidenced by a 2.8-fold decrease in the K M of Mn(II) oxidation. Second, introducing a salt bridge through Lys179Arg mutation improved Glu35 and Glu181 coordination to Mn(II), decreasing K M 2.6-fold. Third, eliminating a steric clash that prevented Glu37 from orienting toward Mn(II) resulted in an 8.6-fold increase in k cat/K M, arising primarily from a 3.6-fold decrease in K M, with a K M value comparable to that of the native enzyme (0.28 mM vs 0.19 mM for Pleurotus eryngii MnP PS3). We further demonstrated that while the effects of Tyr36Phe and Lys179Arg mutations are additive, because involved in secondary-shell interactions to different ligands, other combinations of mutations were antagonistic because they act on different aspects of the Mn(II) coordination at the same residues. Finally, we showed that these MnCcP variants are functional models of MnP that mimic its activity in both Mn(II) oxidation and degradation of a phenolic lignin model compound and kraft lignin. In addition to achieving KM in a designed protein that is similar to the that of native enzyme, our results offer molecular insight into the role of noncovalent interactions around metal-binding sites for improving metal binding and overall activity; such insight can be applied to rationally enhance these properties in other metalloenzymes and their models.
Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the ...nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M–1 cm–1) and 558 nm (2290 M–1 cm–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A ∥ = 98 × 10–4 cm–1, and another more rhombic, with a smaller A ∥ value of 69 × 10–4 cm–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.
Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we ...report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.
In this research, a series of triaxial compression experiments and X-ray observations were conducted to explore the strength, deformability and internal damage mechanism of deeply buried marble. The ...results show that an increase in confining pressure results in obvious brittle–ductile transition characteristics of deeply buried marble. The Young’s modulus of the marble increased nonlinearly with increasing confining pressure. The peak and residual strength of the marble exhibit a clear linear relationship with the confining pressure, which can be described by the linear Mohr–Coulomb criterion. The sensitivity of the residual strength on the confining pressure was clearly higher than that of the peak strength. After uniaxial and triaxial compression failure, marble specimens were analyzed using a three-dimensional X-ray micro-CT scanning system. Based on horizontal and vertical cross-sections, the marble specimen is mainly dominated by axial splitting tensile cracks under uniaxial compression, but under confining pressure, the marble specimen is mainly dominated by a single shear crack. To quantitatively evaluate the internal damage of the marble material, the crack area and aperture extent for each horizontal cross-section were calculated by analyzing the binarized pictures. The system of crack planes under uniaxial compression is more complicated than that under triaxial compression, which is also supported by the evolution of the crack area and aperture extent. Finally, the brittle–ductile transition mechanism of the marble is discussed and interpreted according to the proposed conceptual models.
PDF
