Fluid solution and solid-state electron nuclear double resonance studies of flavin model compounds and flavoenzymes

Kurreck, H.; Bock, Michael; Bretz, Norbert; Elsner, Martin; Kraus, Harold; Lubitz, Wolfgang; Müller, Franz; Geissler, Johanna; Kroneck, Peter M. H.

doi:10.1021/ja00315a048

Cited by 86 publications

(123 citation statements)

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“…The proton hfcs also allow some information on the electronic structure of the cofactor to be drawn. First, the A iso hfc from the methyl H(8␣) protons determined by ENDOR, which is usually considered a guide to the overall spin-density distribution (due to its ease of determination) is one of the smallest yet determined (32,58). This further supports the rather isolated situation of the FMN cofactor in the LOV2 domain and points to a rather nonpolar cofactor-binding site (59).…”

Section: Discussionmentioning

confidence: 59%

Blue Light Perception in Plants

Kay¹,

Schleicher²,

Kuppig³

et al. 2003

Journal of Biological Chemistry

112

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The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420 -480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.

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Section: Discussionmentioning

confidence: 59%

Blue Light Perception in Plants

Kay¹,

Schleicher²,

Kuppig³

et al. 2003

Journal of Biological Chemistry

112

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“…The same effect has been reported for the methanol oxidase semiquinone, a member of the GMC oxidoreductase family which is believed to share a common mechanism with cholesterol oxidase. ENDOR experiments unambiguously demonstrate that, after addition of hydroxylamine (a 'pseudosubstrate') to methanol oxidase, a different FAD radical is generated in the enzyme [14]. Fig.…”

Section: Electron Nuclear Double Resonancementioning

confidence: 79%

“…A second group of signals due to more strongly-coupled protons is observed in the regions 8-10 and 19-21 MHz, corresponding to hyperfine couplings of [9][10][11][12] MHz. Previous work, using isotope-labelling of model flavin semiquinones and flavoenzyme semiquinones, has demonstrated that couplings of this magnitude are dirplayed by 8-CH, protons in both anionic and neutral radicals [14,26,271. Strong couplings also occur to the ring proton on C,.…”

Section: Optical Spectroscopymentioning

confidence: 97%

ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state

Medina

Vrielink

Cammack

1994

European Journal of Biochemistry

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Reduction of the flavin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state. This intermediate had an optical spectrum characteristic of a flavin anionic semiquinone. The rate and extent of reduction were pH-dependent. No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant. No intermediate radical was detected during the reoxidation process. Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state. The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M-' cm-'. The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004. The linewidth of the signal was 1.48 mT when the protein was studied in H,O or D,O. These data are in agreement with those reported for anionic semiquinones. The linewidths were the same when measured either at X-band or at S-band frequencies, indicating that line broadening is due to hyperfine interactions. The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added. Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 9-12 MHz, is attributed to protons on &CH, (A,,, = 10.9 MHz) and on C, (A,so = 9 MHz) of the flavin ring. No change in these hyperfine coupling constants was detected when the protein was studied in D,O. However, the hyperfine coupling constant attributed to protons on 8-CH, decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (A,,, = 9.92 MHz). A second group of signals was observed with hyperfine couplings less than 2.5 MHz. Some of these weak couplings disappeared when the protein was transferred to D,O, or when the substrate, dehydroisoandrosterone, was present. These signals are attributed to displaced water protons, or to exchangeable protons from amino-acid residues on the protein near the flavin binding site, involved in substrate stabilisation.Cholesterol oxidase (3P-hydroxysteroid oxidase, EC 1.1.3.6) is a flavin-dependent enzyme that catalyses the oxidation and isomerisation of 3P-hydroxysteroids having a double bond at A5-A6 of the steroid ring backbone. Although the enzyme exhibits a broad range of steroid specificity, the presence of a 3P-hydroxyl group is essential for activity; 3a-hydroxysteroids are not oxidised by the enzyme [l-31. The highest enzyme activity is observed using cholesterol as a substrate. The enzyme from the soil bacterium Brevibacterium sterolicum is a monomeric oxidase containing one mol...

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“…Such a contact interaction could result from a nitrogen of the flavin ring. The observed hyperfine couplings (^iso of 0.8-2 MHz) are less than those expected for nitrogens at position 5 or 10 of the flavin ring, typically 20 and 9 MHz respectively for an anionic flavin semiquinone [20][21][22]. Couplings of this magnitude are too large for detection by conventional ESEEM [23].…”

Section: Discussionmentioning

confidence: 99%

Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum

1997

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The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA. Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14 N nuclei magnetically coupled to the paramagnet. In 2 Η2θ buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre. Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system. The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.

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Fluid solution and solid-state electron nuclear double resonance studies of flavin model compounds and flavoenzymes

Cited by 86 publications

References 0 publications

Blue Light Perception in Plants

Blue Light Perception in Plants

ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state

Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum

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