Rationally tuning the reduction potential of a single cupredoxin beyond the natural range

Marshall, Nicholas; Garner, Dewain K.; Wilson, Tiffany D.; Gao, Yi; Robinson, Howard; Nilges, Mark J.; Lu, Yi

doi:10.1038/nature08551

Cited by 278 publications

(404 citation statements)

References 29 publications

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“…One possible explanation of the different ET rates is structural perturbation of the Cu-ligand interactions and thus in ET pathways of the different Az mutants. The observed 3D structures (25) and EXAFS data (SI Text) show minimal perturbation of the Cu-ligand distances or angles in the mutants compared with WT Az, which rule out this possibility as the main contributing factor in the variants studied here.…”

Section: Discussionmentioning

confidence: 98%

“…S5). The analysis indicates that the relative conformational freedom has increased, or the rigidity has decreased, particularly around the copper binding site (SI Text and Table S5) (25).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Asn47 was changed to Ser and Phe114 to Asn or Pro to modify the hydrogen bonding networks near Gly45, Cys112, and His117, which interact with the copper. The mutations tuned the reduction potential of the Cu center over a wide range (>500 mV) without significantly disrupting the copper binding site (25), providing a promising system for a quantitative examination of the relationship between ET driving force and rate constant. Here we report the results of studying intramolecular ET from a disulfide radical anion, produced by pulse radiolysis, to the Cu(II) center in several of these Az mutants.…”

Section: Marcus Inverted Regionmentioning

confidence: 99%

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Designed azurins show lower reorganization free energies for intraprotein electron transfer

Farver

Marshall

Wherland

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.cupredoxin | Type 1 copper | blue copper | reorganization energy | Marcus inverted region E lectron transfer (ET) through proteins is central to biological energy conversion processes, from photosynthesis to respiration (1). ET rates between redox centers are tightly controlled and tuned in biological systems for efficiency and to avoid formation of deleterious products (2-5). An important parameter determining the ET rate is the reorganization free energy of the redox sites (6-8), which for biochemical ET reactions has evolved to low values, usually providing high rate constants. Despite many years of work, few efforts have succeeded in designing protein ET sites having reorganization free energies lower than those observed for the native ones. For example, numerous mutations proximal to the type 1 (T1) blue copper site have been introduced into azurin (Az) (9-19), a bacterial electron-mediating protein, but those that have lowered this barrier have not been reported yet.Prominent among the theories developed to rationalize the control of ET reaction rates (20-22) is that of R. A. Marcus (23). A particularly intriguing element of this theory predicts that the ET rate reaches its maximum when the driving force of the reaction equals the reorganization free energy, after which an even higher driving force decreases ET rate, reaching the "Marcus inverted region." It has been proposed that the dramatic difference in rates between some of the forward and backward ET reactions in the photosynthetic reaction center is a result of the latter being in the inverted region, which is reached because of exceptionally low reorganization free energies of the relevant ET sites (24).In the present study, we aim at examining how the major changes in modifying the redox potential of the type 1 copper site in Az achieved by Marshall et al. (25) affect its ET reactivity. A major obstacle in rationally fine-tuning the properties of t...

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Section: Discussionmentioning

confidence: 98%

“…S5). The analysis indicates that the relative conformational freedom has increased, or the rigidity has decreased, particularly around the copper binding site (SI Text and Table S5) (25).…”

Section: Discussionmentioning

confidence: 99%

Section: Marcus Inverted Regionmentioning

confidence: 99%

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Designed azurins show lower reorganization free energies for intraprotein electron transfer

Farver

Marshall

Wherland

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…AsCBP1 encodes a cupredoxin superfamily protein. Proteins from this family function in oxidation homeostasis and electron transfer reactions, which are involved in photosynthesis, respiration, cell signaling, and numerous reactions of oxidases and reductases (Lu et al, 2004;Solomon et al, 2004;Dennison, 2005;Marshall et al, 2009).…”

Section: Osa-mir528 Putative Target Identification and Responses To Smentioning

confidence: 99%

Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass

et al. 2015

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MicroRNA528 (miR528) is a conserved monocot-specific small RNA that has the potential of mediating multiple stress responses. So far, however, experimental functional studies of miR528 are lacking. Here, we report that overexpression of a rice (Oryza sativa) miR528 (Osa-miR528) in transgenic creeping bentgrass (Agrostis stolonifera) alters plant development and improves plant salt stress and nitrogen (N) deficiency tolerance. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller number, and upright growth. Improved salt stress resistance is associated with increased water retention, cell membrane integrity, chlorophyll content, capacity for maintaining potassium homeostasis, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while enhanced tolerance to N deficiency is associated with increased biomass, total N accumulation and chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. In addition, AsAAO and COPPER ION BINDING PROTEIN1 are identified as two putative targets of miR528 in creeping bentgrass. Both of them respond to salinity and N starvation and are significantly down-regulated in miR528-overexpressing transgenics. Our data establish a key role that miR528 plays in modulating plant growth and development and in the plant response to salinity and N deficiency and indicate the potential of manipulating miR528 in improving plant abiotic stress resistance.Abiotic stresses, especially drought, salt, and nitrogen (N) deficiency, are limiting factors for plant growth, development, and agricultural productivity. To cope with drought and salt stresses, plants have evolved similar strategies of osmotic adjustment (Munns, 2002;Zhu, 2002). Plants also evolved salinity-specific adjustments against ionic disequilibrium, which encompass excluding salt entry into plants and compartmentalizing ions into vacuoles or old leaves (Munns, 1993;Yeo, 1998). Another worldwide limiting factor for crop yields is N deficiency, which triggers reduced leaf growth rate and photosynthetic rate (Chapin et al., 1988). Due to the sessile nature of plants, abiotic stresses are unavoidable. Therefore, it is critical to develop reliable procedures to genetically modify plants for improved performance under environmental stresses, thereby enhancing agricultural productivity to meet the ever-growing demands in food production.Genetic engineering plays an increasingly important role in agronomic trait modifications in crop species. Currently, many genes encoding functional proteins, transcription factors, and proteins involved in signaling pathways have been identified as abiotic stressresponsive genes (Shinozaki and Yamaguchi-Shinozaki, 2007;Masclaux-Daubresse et al., 2010;Turan et al., 2012). Constitutive expression of some of these genes in transgenic plants has been demonstrated to lead to enhanced salt or drought tolerance (Golldack et al., 2011;Kim et al., 2013;Lu et al., 2013;Li et al., 2014). However, details of the regulatory network in the plant...

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“…In an earlier study, Marshall et al found that the redox potential of some metalloproteins changed when an amino acid close to the active site was replaced. 32 In Nii3-WT and Nii4-WT, Residue 448 is located close to the [4Fe-4S] cluster (Fig. 6).…”

Section: Function Of the Met175 Residue In Nii3-wtmentioning

confidence: 99%

Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high‐resolution structures

et al. 2012

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Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3), and root-type (Nii2 and Nii4). The high-resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E, and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (k cat /K m ). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k cat ) and Michaelis constant (K m ) of Nii4-WT were lower than those of Nii3-WT. However, the k cat /K m of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change in k cat /K m was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.

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Rationally tuning the reduction potential of a single cupredoxin beyond the natural range

Cited by 278 publications

References 29 publications

Designed azurins show lower reorganization free energies for intraprotein electron transfer

Designed azurins show lower reorganization free energies for intraprotein electron transfer

Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass

Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high‐resolution structures

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