One- and Two-Dimensional ESEEM Spectroscopy of Flavoproteins

Martínez, Jesús I.; Alonso, Pablo J.; Gómez‐Moreno, Carlos; Medina, Milagros

doi:10.1021/bi971495g

Cited by 34 publications

(70 citation statements)

References 42 publications

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“…[28] For these two species, the analysis of the correlation ridges yielded nearly identical hfcs of axial symmetry with A k = (À1.4 AE 0.9) MHz and A ? = (À27.5 AE 0.7) MHz (flavodoxin) and A k = (À2.3 AE 1.1) MHz and A ?…”

Section: X-band Pulsed (Davies) Endor Spectroscopymentioning

confidence: 88%

Probing the N(5)H Bond of the Isoalloxazine Moiety of Flavin Radicals by X‐ and W‐Band Pulsed Electron–Nuclear Double Resonance

et al. 2005

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An X- (9.7 GHz and W-band (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T = 80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chiD/chiH, reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-exchanged sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R(-3) dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences can be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.

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Section: X-band Pulsed (Davies) Endor Spectroscopymentioning

confidence: 88%

Probing the N(5)H Bond of the Isoalloxazine Moiety of Flavin Radicals by X‐ and W‐Band Pulsed Electron–Nuclear Double Resonance

et al. 2005

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“…Recent ab initio theoretical calculations showed that most of the spin density in anionic flavin semiquinones is located either on the N (5) position or the benzene moiety of the isoalloxazine ring (38,39). The N (5) locus is expected to be freely accessible to oxygen, making it a good candidate for reaction with oxygen in 2-nitropropane dioxygenase via a rapid one-electron transfer that results in the oxidation of the flavin and formation of superoxide (Scheme 4).…”

Section: Discussionmentioning

confidence: 99%

Involvement of a Flavosemiquinone in the Enzymatic Oxidation of Nitroalkanes Catalyzed by 2-Nitropropane Dioxygenase

Francis

Russell

Gadda

2005

Journal of Biological Chemistry

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2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1-and propyl-2-nitronate, for which a nonenzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k cat /K m determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[ 2 H 4 ]nitroethane and 1,1,2-[ 2 H 3 ]ethyl nitronate at pH 8 yielded normal and inverse effects on the k cat /K m value, respectively, and were negligible on the k cat value. The k cat /K m and k cat pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.2-Nitropropane dioxygenase (EC 1.13.11.32) from Neurospora crassa is a flavin-dependent enzyme, which catalyzes the oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrite. The enzyme was first characterized by Little (1) in 1951 as an oxidase, but was later classified as a dioxygenase based on the observation that the oxygen atom of the organic product formed upon oxidation of propyl-2-nitronate originates from molecular oxygen and not from water (2). To date, 2-nitropropane dioxygenase has been isolated from N. crassa (1) and Hansenula mrakii (3). Whereas the two enzymes have similar molecular weights of about 40,000, they differ in their prosthetic group content in that...

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“…ϩ e Ϫi⅐2͑␣t2Ϫ␤t1ϩ͑␣Ϫ␤͒ / 2͒ ϩ c.c.͒͘ ⍀ , [11] where c.c. indicates the complex conjugate of the previous terms, and ͗..͘ ⍀ the powder average over different orientations ⍀ of the external magnetic field.…”

Section: Interference Effects In Powder Hyscore Spectramentioning

confidence: 99%

“…One can consider only the first term e Ϫi⅐2(␣ t1 ϩ␤ t2 ϩ(␣ ϩ␤ ) / 2) of Eq. [11] without loss of generality, because each term produces a distinct peak that nevertheless is subject to similar considerations.…”

Section: Interference Effects In Powder Hyscore Spectramentioning

confidence: 99%