Isolation and characterization of the flavin-binding domain of flavocytochrome <i>b</i>2 expressed independently in <i>Escherichia coli</i>

Balme, Alexis; Brunt, Claire E; Pallister, R L; Chapman, Stephen K; Reid, Graeme A.

doi:10.1042/bj3090601

Cited by 25 publications

(42 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3-N-methyllumiflavin (3mLF), prepared according to Yoneda et al (9), was a gift from Dr. H. I. X. Mager, Department of Biochemical and Biophysical Sciences, University of Houston. The FDH, a gift from Dr. F. Lederer, Laboratoire d'Enzymologie, Gif-sur-Yvette, France, was obtained as described by Balme et al (10). (The structure of flavin is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Excited‐State Dynamics of Fully Reduced Flavins and Flavoenzymes Studied at Subpicosecond Time Resolution

Enescu

Lindqvist

Soep

1998

Photochem & Photobiology

View full text Add to dashboard Cite

The photophysics of the fully reduced states of a number of flavins (flavin mononucleotide, flavin adenine dinucleotide and 3-N-methyllumiflavin) and flavoenzymes (glucose oxidase from Aspergillus niger and the flavodehydrogenase component isolated from flavocytochrome b2) was studied using subpicosecond laser excitation at lambda = 312 nm. The prompt transient absorption spectra (measured from 400 to 850 nm) were all closely similar in the case of the free flavins in aqueous solution. The decay of the transient absorbance obeyed biexponential kinetics with a fast component of lifetime ranging from 4 to 130 ps and a slower phase with a lifetime above 1 ns. The spectral structure changed appreciably during the rapid decay phase. In contrast, in the case of the enzymes only a very slight decay was apparent over the probed time interval (1 ns) and the shape of the spectrum remained unchanged. It is proposed that the two transient spectra appearing in the free flavins correspond to two conformations differing by their degree of nonplanarity, whereas in the flavoenzymes only one conformation is possible.

show abstract

Section: Methodsmentioning

confidence: 99%

Excited‐State Dynamics of Fully Reduced Flavins and Flavoenzymes Studied at Subpicosecond Time Resolution

Enescu

Lindqvist

Soep

1998

Photochem & Photobiology

View full text Add to dashboard Cite

show abstract

“…This domain is homologous to the other members of this flavoprotein family and is capable of converting lactate to pyruvate by itself. [42][43][44] The first 100 residues of the protein contain a heme which accepts electrons from the reduced flavin and transfers them to an acceptor. We have been using a combination of isotope effects and mutagenesis to probe the mechanism of lactate oxidation by yeast flavocytochrome b 2 .…”

Section: A-hydroxy Acid Oxidases and Dehydrogenasesmentioning

confidence: 99%

Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Fitzpatrick

2007

Labelled Comp Radiopharmac

View full text Add to dashboard Cite

Deuterium, solvent, and (15)N kinetic isotope effects have been used to probe the mechanisms by which flavoproteins oxidize carbon-oxygen and carbon-nitrogen bonds in amines, hydroxy acids, and alcohols. For the amine oxidases d-amino acid oxidase, N-methyltryptophan oxidase, and tryptophan monooxygenase, d-serine, sarcosine, and alanine are slow substrates for which CH bond cleavage is fully rate limiting. Inverse isotope effects for each of 0.992-0.996 are consistent with a common mechanism involving hydride transfer from the uncharged amine. Computational analyses of possible mechanisms support this conclusion. Deuterium and solvent isotope effects with wild-type and mutant variants of the lactate dehydrogenase flavocytochrome b(2) show that OH and CH bond cleavage are not concerted, but become so in the Y254F enzyme. This is consistent with a highly asynchronous reaction in which OH bond cleavage precedes hydride transfer. The results of Hammett analyses and solvent and deuterium isotope effects support a similar mechanism for alcohol oxidase.

show abstract

“…The reason for this biphasic behaviour in the wild-type enzyme lies in the fact that the tetramer requires 12 electrons to become fully reduced. After the initial fast reduction of the four FMN groups by four molecules of lactate, there is a redistribution of the electrons to reduce the heme moieties, followed by complete reduction of the tetramer by a further two molecules of lactate in the slow phase of the reaction [22]. However, in a system such as H66C-b 2 where the heme is not involved, this slower second phase cannot occur and monophasic kinetics would be expected.…”

Section: Spectral Characteristicsmentioning

confidence: 99%

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

et al. 2000

View full text Add to dashboard Cite

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

show abstract

Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli

Cited by 25 publications

References 22 publications

Excited‐State Dynamics of Fully Reduced Flavins and Flavoenzymes Studied at Subpicosecond Time Resolution

Excited‐State Dynamics of Fully Reduced Flavins and Flavoenzymes Studied at Subpicosecond Time Resolution

Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Contact Info

Product

Resources

About