| 1. |
- Berggren, Gustav, et al.
(författare)
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15.02 - Hydrogenases and Model Complexes in Bioorganometallic Chemistry
- 2022
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Ingår i: Comprehensive Organometallic Chemistry IV. - Oxford : Elsevier. - 9780323913508 ; , s. 3-40
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Hydrogenases are enzymes involved in H2 metabolism, and provide a blue-print for how efficient H2/H+ interconversion can be achieved utilizing base metals. The societal interest in H2 as a future energy carrier, and a desire for fundamental understanding of how their biologically unique cofactors operate, has promoted intense studies of these enzymes and their related biomimetic analogs. This book chapter provides an overview of both the biochemistry of these fascinating enzymes as well the extensive work related to preparing and characterizing organometallic complexes mimicking their catalytic cofactors.
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| 2. |
- Berglund, Sigrid, et al.
(författare)
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Hydrogen production by a fully de novo enzyme
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterised by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo protein produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with turnover numbers around 80% of that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, how our study contributes important information about the functionality of cobaloxime as hydrogen evolving catalysts in protein environments, and the feasibility of using de novo proteins for developent into artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscalable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that these types of novel enzymes are a possible product.
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| 3. |
- Glover, Starla, et al.
(författare)
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A Quick and Colorful Method to Measure Low-Level Contaminations of Paramagnetic Ni2+ in Protein Samples Purified by Immobilized Metal Ion Affinity Chromatography
- 2019
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Ingår i: BIOLOGICAL NMR PT A. - : ELSEVIER ACADEMIC PRESS INC. - 9780128138601 ; , s. 87-106
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Bokkapitel (refereegranskat)abstract
- Isotopic labeling of recombinantly expressed proteins is generally required for investigation by modern nuclear magnetic resonance (NMR) methods. Purification strategies of the labeled proteins often include the use of a polyhistidine affinity tag (His-tag) and immobilized metal ion affinity chromatography (IMAC). Described herein are rapid and inexpensive qualitative and quantitative assays to determine the concentration of paramagnetic Ni2+ in protein samples purified by IMAC. Both qualitative and quantitative colorimetric methods detect the amount of Ni2+ via the color change produced when a [Ni(PAR)(n)](2+) (PAR = 4-(2-pyridylazo)resorcinol, n = 1, 2) complex is formed. The qualitative assay provides a rapid visual test for the presence of Ni2+ in the low micromolar range in a sample of interest. The usefulness of the spectroscopic quantitative assay is illustrated by: (i) detecting a 12 mu M Ni2+ contamination in an NMR sample containing 950 mu M of the 7.5kDa alpha W-3 protein purified by a standard His-tag Ni2+/IMAC approach and (ii) showing that the N-15-HSQC spectrum of the alpha W-3 NMR sample, containing 1 paramagnetic Ni2+ ion per 80 protein molecules, displays clear line broadening of both water and protein spectral lines. We also (iii) measured Ni2+ release during the equilibration, wash, and elution steps of three commonly used Ni2+/IMAC resins when following manufacturer's protocols. The concentration of Ni2+ detected in elutes of the three resins ranged from 2 mu M to nearly 1mM.
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| 4. |
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| 5. |
- Glover, Starla D., et al.
(författare)
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Isolating the Effects of the Proton Tunneling Distance on Proton-Coupled Electron Transfer in a Series of Homologous Tyrosine-Base Model Compounds
- 2017
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Ingår i: Journal of the American Chemical Society. - : AMER CHEMICAL SOC. - 0002-7863 .- 1520-5126. ; 139:5, s. 2090-2101
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Tidskriftsartikel (refereegranskat)abstract
- The distance dependence of concerted proton-coupled electron transfer (PCET) reactions was probed in a series of three new compounds, where a phenol is covalently bridged by a 5, 6, or 7 membered carbocycle to the quinoline. The carbocycle bridge enforces the change in distance between the phenol oxygen (proton donor) and quinoline nitrogen (proton acceptor), d(O center dot center dot center dot N), giving rise to values ranging from 2.567 to 2.8487 angstrom, and resulting in calculated proton tunneling distances, r(0), that span 0.719 to 1.244 angstrom. Not only does this series significantly extend the range of distances that has been previously accessible for experimental distance dependent PCET studies of synthetic model compounds, but it also greatly improves the isolation of d(O center dot center dot center dot N) as a variable compared to earlier reports. Rates of PCET were determined by time-resolved optical spectroscopy with flash-quench generated [Ru(bpy)(3)](3+) and [Ru(dce)(3)](3+), where bpy = 2,2'-bipyridyl and dce = 4,4'-dicarboxyethylester-2,2'-bipyridyl. The rates increased as d(O center dot center dot center dot N) decreased, as can be expected from a static proton tunneling model. An exponential attenuation of the PCET rate constant was found: k(PCET)(d) = k(PCET)(0)exp[-beta(d-d(0))], with beta similar to 10 angstrom(-1). The observed kinetic isotope effect (KIE = k(H)/k(D)) ranged from 1.2 to 1.4, where the KIE was observed to decrease slightly with increasing d(O center dot center dot center dot N). Both beta and KIE values are significantly smaller than what is predicted by a static proton tunneling model. We conclude that vibrational compression of the tunneling distances, as well as higher vibronic transitions, that contribute to concerted proton coupled electron transfer must also be considered.
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| 6. |
- Glover, Starla D., et al.
(författare)
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Photochemical Tyrosine Oxidation in the Structurally Well-Defined alpha Y-3 Protein : Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136:40, s. 14039-14051
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Tidskriftsartikel (refereegranskat)abstract
- Tyrosine oxidation-reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The alpha Y-3 model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of alpha Y-3 are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in alpha Y-3. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2'-bipyridine)(3)](3+) oxidant provides high-quality Y32-O center dot absorption spectra. The rate constant of Y32 oxidation (k(pCET)) is pH dependent: 1.4 x 10(4) M-1 s(-1) (pH 5.5), 1.8 x 10(5) M-1 s(-1) (pH 8.5), 5.4 x 10(3) M-1 s(-1) (pD 5.5), and 4.0 x 10(4) M-1 s(-1) (pD 8.5). k(H)/k(D) of Y32 oxidation is 2.5 +/- 0.5 and 4.5 +/- 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32-O center dot is 28-58% versus the concentration of [Ru(2,2'-bipyridine)(3)](3+). Y32-O center dot decays slowly, t(1/2) in the range of 2-10 s, at both pH 5.5 and 8.5, via radical-radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32-O center dot is discussed relative to the structural properties of the Y32 site. Finally, the static alpha Y-3 NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32-O center dot come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of alpha Y-3.
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| 7. |
- Glover, Starla, et al.
(författare)
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Pourbaix Diagram, Proton-Coupled Electron Transfer, and Decay Kinetics of a Protein Tryptophan Radical : Comparing the Redox Properties of W32• and Y32• Generated Inside the Structurally Characterized α3W and α3Y Proteins
- 2018
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:1, s. 185-192
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Tidskriftsartikel (refereegranskat)abstract
- Protein-based “hole” hopping typically involves spatially arranged redox-active tryptophan or tyrosine residues. Thermodynamic information is scarce for this type of process. The well-structured α3W model protein was studied by protein film square wave voltammetry and transient absorption spectroscopy to obtain a comprehensive thermodynamic and kinetic description of a buried tryptophan residue. A Pourbaix diagram, correlating thermodynamic potentials (E°′) with pH, is reported for W32 in α3W and compared to equivalent data recently presented for Y32 in α3Y (Ravichandran, K. R.; Zong, A. B.; Taguchi, A. T.; Nocera, D. G.; Stubbe, J.; Tommos, C. J. Am. Chem. Soc. 2017, 139, 2994−3004). The α3W Pourbaix diagram displays a pKOX of 3.4, a E°′(W32(N•+/NH)) of 1293 mV, and a E°′(W32(N•/NH); pH 7.0) of 1095 ± 4 mV versus the normal hydrogen electrode. W32(N•/NH) is 109 ± 4 mV more oxidizing than Y32(O•/OH) at pH 5.4–10. In the voltammetry measurements, W32 oxidation–reduction occurs on a time scale of about 4 ms and is coupled to the release and subsequent uptake of one full proton to and from bulk. Kinetic analysis further shows that W32 oxidation likely involves pre-equilibrium electron transfer followed by proton transfer to a water or small water cluster as the primary acceptor. A well-resolved absorption spectrum of W32• is presented, and analysis of decay kinetics show that W32• persists ∼104 times longer than aqueous W• due to significant stabilization by the protein. The redox characteristics of W32 and Y32 are discussed relative to global and local protein properties.
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| 8. |
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| 9. |
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| 10. |
- Nilsen-Moe, Astrid, et al.
(författare)
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Concerted and Stepwise Proton-Coupled Electron Transfer for Tryptophan-Derivative Oxidation with Water as the Primary Proton Acceptor : Clarifying a Controversy
- 2022
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:16, s. 7308-7319
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Tidskriftsartikel (refereegranskat)abstract
- Concerted electron-proton transfer (CEPT) reactions avoid charged intermediates and may be energetically favorable for redox and radical-transfer reactions in natural and synthetic systems. Tryptophan (W) often partakes in radical-transfer chains in nature but has been proposed to only undergo sequential electron transfer followed by proton transfer when water is the primary proton acceptor. Nevertheless, our group has shown that oxidation of freely solvated tyrosine and W often exhibit weakly pH-dependent proton-coupled electron transfer (PCET) rate constants with moderate kinetic isotope effects (KIE approximate to 2-5), which could be associated with a CEPT mechanism. These results and conclusions have been questioned. Here, we present PCET rate constants for W derivatives with oxidized Ru- and Zn-porphyrin photosensitizers, extracted from laser flash-quench studies. Alternative quenching/photo-oxidation methods were used to avoid complications of previous studies, and both the amine and carboxylic acid groups of W were protected to make the indole the only deprotonable group. With a suitably tuned oxidant strength, we found an ET-limited reaction at pH < 4 and weakly pH-dependent rates at pH > similar to 5 that are intrinsic to the PCET of the indole group with water (H2O) as the proton acceptor. The observed rate constants are up to more than 100 times higher than those measured for initial electron transfer, excluding the electron-first mechanism. Instead, the reaction can be attributed to CEPT. These conclusions are important for our view of CEPT in water and of PCET-mediated radical reactions with solvent-exposed tryptophan in natural systems.
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