Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer

Ohler, Amanda; Long, Hanna; Ohgo, Kei; Tyson, Kristin; Murray, David K.; Davis, Amanda; Whittington, Chris; Hvastkovs, Eli G.; Duffy, Liam M.; Haddy, Alice; Sargent, Andrew L.; Allen, William E.; Offenbacher, Adam R.

doi:10.1039/d1cc00187f

Cited by 3 publications

(11 citation statements)

References 24 publications

(29 reference statements)

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“…EPR parameters are the same for both spectra as given for Figure 2b compared to the indole N deprotonation. This agrees with the results by Ohler et al (2021). For the OxDC active site model, we computed this preference to be even stronger (energy difference of 21 kcal mol À1 ).…”

Section: Dft Calculationssupporting

confidence: 90%

“…Incorporation of tyrosine analogs are well documented and have been used to probe long‐range electron transfer (LRET) in ribonucleotide reductase and cytochrome c peroxidase (Chang, Yee, et al, 2004; Ravichandran et al, 2013; Ravichandran et al, 2017; Reece & Seyedsayamdost, 2017; Yee et al, 2019). Tryptophan analogs are increasingly used in recombinant proteins, primarily for their cross‐linking ability, their fluorescence properties, their ability to serve as pH sensors, and for GCE methods development (Bae et al, 2003; Boknevitz et al, 2019; Buddha & Crane, 2005; Budisa et al, 2002; Englert et al, 2015; Hilaire et al, 2017; Italia et al, 2017; Ohler et al, 2021; Pastore et al, 2022; Pratt & Ho, 1975; Ross et al, 1992; Singh‐Blom et al, 2014; Wong & Eftink, 1997). The selective incorporation of tryptophan analogs became feasible recently by applying GCE technologies as demonstrated by Italia et al (2017), Englert et al (2015), and Ficaretta et al (2022) and is ideal for studying the biophysical role of tryptophan residues because their respective indole side chains possess different electronic properties (Ohler et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

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Selective incorporation of 5‐hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

et al. 2022

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Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.

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Section: Dft Calculationssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Selective incorporation of 5‐hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

et al. 2022

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“…This peptide was designed to mimic the staggered π stacking between W48 and F110 in azurin (Figure A). The electrochemical potential of the β-W2 was found to be 989 ± 9 mV at pH 7.0; this result is comparable to the potential of the tryptophan model, N -acetyl- l -tryptophan ethyl ester, NATE, E net = 983 mV, suggesting that the solvation surrounding the tryptophan impacts the potential. Similarly, a series of tyrosine-containing β-hairpins exhibited electrochemical potentials comparable to that of the side chain alone.…”

Section: Resultsmentioning

confidence: 59%

“…The 12-amino acid peptide, β-W2 (RWVEVNGOKIFQ), was synthesized previously. 28 Circular Dichroism (CD) Spectroscopy. The impact of the mutations on the azurin secondary structure was assessed using CD measurements (Jasco CD spectrometer J-815).…”

Section: ■ Methodsmentioning

confidence: 99%

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Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48

et al. 2022

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Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (E°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode�considered a vibrational marker for hydrogen bonding�and E°is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/ frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E°and reveal properties of the protein microenvironment surrounding tryptophan.

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Expanding the Genetic Code of Bioelectrocatalysis and Biomaterials

Chemla,

Kaufman,

Amiram

et al. 2024

Chem. Rev.

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Genetic code expansion is a promising genetic engineering technology that incorporates noncanonical amino acids into proteins alongside the natural set of 20 amino acids. This enables the precise encoding of non-natural chemical groups in proteins. This review focuses on the applications of genetic code expansion in bioelectrocatalysis and biomaterials. In bioelectrocatalysis, this technique enhances the efficiency and selectivity of bioelectrocatalysts for use in sensors, biofuel cells, and enzymatic electrodes. In biomaterials, incorporating non-natural chemical groups into protein-based polymers facilitates the modification, finetuning, or the engineering of new biomaterial properties. The review provides an overview of relevant technologies, discusses applications, and highlights achievements, challenges, and prospects in these fields.

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Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer

Abstract: Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into β-hairpin peptides and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function...

Cited by 3 publications

References 24 publications

Selective incorporation of 5‐hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Selective incorporation of 5‐hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48

Expanding the Genetic Code of Bioelectrocatalysis and Biomaterials

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