Spectroscopic investigation of flavoproteins: Mechanistic differences between (electro)chemical and photochemical reduction and oxidation

Nöll, Gilbert

doi:10.1016/j.jphotochem.2008.06.019

Cited by 16 publications

(9 citation statements)

References 53 publications

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“…The UV−vis spectrum of the species after electrolysis at pH 7−11 resembles the UV−vis spectrum reported for FMNH − . 24 This not only substantiates the conclusion that the final products at pH 3−5 and 7−11 are FMNH 2 and FMNH − , respectively, but helps to narrow the range for the pK a of FMNH 2 to be between 5 and 7. Indeed, data collected by Hemmerich et al indicated that the pK a of FMNH 2 is around 6.2.…”

Section: Resultssupporting

confidence: 71%

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Differences in Proton-Coupled Electron-Transfer Reactions of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) between Buffered and Unbuffered Aqueous Solutions

2013

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The electrochemical reduction mechanisms of flavin mononucleotide (FMN) in buffered aqueous solutions at pH 3-11 and unbuffered aqueous solutions at pH 2-11 were examined in detail using variable-scan-rate cyclic voltammetry (ν = 0.1-20 V s(-1)), controlled-potential bulk electrolysis, UV-vis spectroscopy, and rotating-disk-electrode voltammetry. In buffered solutions at pH 3-5, FMN undergoes a two-electron/two-proton (2e(-)/2H(+)) reduction to form FMNH2 at all scan rates. When the buffered pH is increased to 7-9, FMN undergoes a 2e(-) reduction to form FMN(2-), which initially undergoes hydrogen bonding with water molecules, followed by protonation to form FMNH(-). At a low voltammetric scan rate of 0.1 V s(-1), the protonation reaction has sufficient time to take place. However, at a higher scan rate of 20 V s(-1), the proton-transfer reaction is outrun, and upon reversal of the scan direction, less of the FMNH(-) is available for oxidation, causing its oxidation peak to decrease in magnitude. In unbuffered aqueous solutions, three major voltammetric waves were observed in different pH ranges. At low pH in unbuffered solutions, where [H(+)] ≥ [FMN], (FMN)H(-) undergoes a 2e(-)/2H(+) reduction to form (FMNH2)H(-) (wave 1), similar to the mechanism in buffered aqueous solutions at low pH. At midrange pH values (unbuffered), where pH ≤ pKa of the phosphate group and [FMN] ≥ [H(+)], (FMN)H(-) undergoes a 2e(-) reduction to form (FMN(2-))H(-) (wave 2), similar to the mechanism in buffered aqueous solutions at high pH. At high pH (unbuffered), where pH ≥ pKa = 6.2 of the phosphate group, the phosphate group loses its second proton to be fully deprotonated, forming (FMN)(2-), and this species undergoes a 2e(-) reduction to form (FMN(2-))(2-) (wave 3).

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Section: Resultssupporting

confidence: 71%

“…24 The intense colors observed during the bulk reduction suggest that some of the intermediate radical compounds might have a finite lifetime (possibly due to homogeneous reactions between the highly reduced FMNH 2 and the starting material) before all are ultimately converted to FMNH 2 as the electrolysis progresses.…”

Section: Resultsmentioning

confidence: 99%

Differences in Proton-Coupled Electron-Transfer Reactions of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) between Buffered and Unbuffered Aqueous Solutions

2013

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“…Dithionite-reduced HoxFU samples display shoulders at 553 nm, 605 nm and 665 nm ( Figure 3 , panel B), which are found for neutral semiquinone radicals [31] , [32] , [33] , [34] , [35] , [36] . We cannot exclude contributions from [Fe-S] clusters to these signals.…”

Section: Resultsmentioning

confidence: 99%

“…However, due to the low extinction coefficients of [2Fe-2S] and [4Fe-4S] clusters in the reduced state, they should be minor. The additional signals at 370 nm 388 nm and 399 nm can be attributed to the anionic semiquinone radical form of the flavin [31] , [34] , [35] , [36] . So far the anionic semiquinone level of the flavin has not been reported for the native SH from R. opacus or R. eutropha [17] suggesting that this state is only formed at low potentials that can be experienced in isolated HoxFU.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

et al. 2011

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The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.

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“…In line with the extreme versatility of voltammetry in its variant modes (linear sweep,c yclic, or hydrodynamic) for the electrochemical study of organic molecules, [57] this technique was primarily employed in the presents tudy on 1-4 ( Figure 1) in conjunction with UV/Vis spectroelectrochemical (SPEC) experi-ments [36,40,59] for mechanistice lucidationsr elatedt op rotoncoupled electron transfer (PCET). Important aspects related to PCET such as electron/proton stoichiometry and its sensitivity to solution pH, and proton starvation effects at the electrodesolution interface are discussed below,a si st he influence of Nalkyl substitution (at N1/N10 and N3) on the redox potential of 1-4.F inally,apreliminarya ssessment of the electrocatalytic properties of 1 for the dioxygen reductionr eaction( ORR) is also presented.…”

Section: Introductionmentioning

confidence: 99%

Flavin Derivatives with Tailored Redox Properties: Synthesis, Characterization, and Electrochemical Behavior

Kormányos

Hossain

Ghadimkhani

et al. 2016

Chemistry A European J

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This study establishes structure-property relationships for four synthetic flavin molecules as bioinspired redox mediators in electro- and photocatalysis applications. The studied flavin compounds were disubstituted with polar substituents at the N1 and N3 positions (alloxazine) or at the N3 and N10 positions (isoalloxazines). The electrochemical behavior of one such synthetic flavin analogue was examined in detail in aqueous solutions of varying pH in the range from 1 to 10. Cyclic voltammetry, used in conjunction with hydrodynamic (rotating disk electrode) voltammetry, showed quasi-reversible behavior consistent with freely diffusing molecules and an overall global 2e(-) , 2H(+) proton-coupled electron transfer scheme. UV/Vis spectroelectrochemical data was also employed to study the pH-dependent electrochemical behavior of this derivative. Substituent effects on the redox behavior were compared and contrasted for all the four compounds, and visualized within a scatter plot framework to afford comparison with prior knowledge on mostly natural flavins in aqueous media. Finally, a preliminary assessment of one of the synthetic flavins was performed of its electrocatalytic activity toward dioxygen reduction as a prelude to further (quantitative) studies of both freely diffusing and tethered molecules on various electrode surfaces.

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Spectroscopic investigation of flavoproteins: Mechanistic differences between (electro)chemical and photochemical reduction and oxidation

Cited by 16 publications

References 53 publications

Differences in Proton-Coupled Electron-Transfer Reactions of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) between Buffered and Unbuffered Aqueous Solutions

Differences in Proton-Coupled Electron-Transfer Reactions of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) between Buffered and Unbuffered Aqueous Solutions

Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

Flavin Derivatives with Tailored Redox Properties: Synthesis, Characterization, and Electrochemical Behavior

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