Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

Kok, A. de; Visser, Antonie J. W. G.

doi:10.1016/0014-5793(87)81033-5

Cited by 21 publications

(12 citation statements)

References 14 publications

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“…Flavins, such as FAD, are endogenous redox cofactors that are fluorescent in the oxidized state, so in principle should provide an inverse readout from NAD(P)H (7). However, the fluorescence properties of flavins are heavily modified by the enzymes within which they are contained, and the majority of FPs, including those incorporated in the RC, are not thought to be fluorescent (10). Therefore, it is possible that visible FP signals could be functionally related more to specific metabolic pathways, rather than the redox state of the RC.…”

Section: Discussionmentioning

confidence: 99%

“…NADPH has an important role in the synthesis of macromolecules and in maintaining antioxidant defenses and has identical excitation/ emission wavelengths to NADH (5), thus the two signals are often grouped together [NAD(P)H]. Flavins, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide, are important redox cofactors that emit in the green range (520 -560 nm) when in an oxidized state (7), and their fluorescence properties depend on the FP within which they are contained (10). Previous studies of mitochondria isolated from the kidney have suggested that the main sources of flavin fluorescence are the FAD-containing electron transfer FP (ETF), which feeds electrons from beta oxidation into the RC, and lipoamide dehydrogenase (LipDH), a component of the enzymes pyruvate-dehydrogenase (PDH) and ␣-ketoglutarate-dehydrogenase (also known as oxoglutarate dehydrogenase), which are involved in pyruvate and glutamine metabolism, respectively (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

Bugarski

Martins

Haenni

et al. 2018

American Journal of Physiology-Renal Physiology

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Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that auto-fluorescence signals provide important functional readouts of redox state and substrate metabolism, and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both S1 and S2 PT segments, and was sensitive to changes in respiratory chain (RC) redox state, while cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate, but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate, and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1, but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate, but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

Bugarski

Martins

Haenni

et al. 2018

American Journal of Physiology-Renal Physiology

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“…Red-edge spectroscopy in conjunction with temperature variation provides the necessary experimental axes to assess diagnostic methods to discriminate between reorientational dynamics and energy transfer. We can therefore exclude restricted motion ofthe flavins or hinge motion of the subunits as alternative mechanisms causing depolarization of the fluorescence (de Kok and Visser, 1987).…”

Section: Discussionmentioning

confidence: 99%

“…The intrinsically fluorescent prosthetic group ofmost ofthese proteins is either flavin adenine dinucleotide (FAD)' or flavin mononucleotide, which both have the spectroscopically relevant isoalloxazine in common. The fluorescence properties of the isoalloxazinic ring vary largely among different flavoproteins, reflecting the variety in structure and dynamical properties of the active sites containing the flavin compounds (Visser et al, 1974(Visser et al, , 1980de Kok and Visser, 1987;Bastiaens et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study

Bastiaens¹,

Hoek²,

Benen³

et al. 1992

Biophysical Journal

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Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.

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“…The limited number of publications on 2P-microscopy imaging of cellular flavin reflect its experimental limitations, namely a low excitation cross-section and an abysmal fluorescence quantum yield (0.03) [148], which is attributed to dynamic quenching by adenine [148,149], van der Waals interactions with tryptophan residues and/or the sulfur atoms in cysteine residues [150,151]. The actual concentrations and distribution of FAD in live cells remains uncertain, especially under different physiological conditions.…”

Section: Experimental Approaches To Interrogate Intracellular Coenzymesmentioning

confidence: 99%

Intracellular Coenzymes as Natural Biomarkers for Metabolic Activities and Mitochondrial Anomalies

2010

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Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation–reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.

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Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

Cited by 21 publications

References 14 publications

Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study

Intracellular Coenzymes as Natural Biomarkers for Metabolic Activities and Mitochondrial Anomalies

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