Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+-Reductase

Serrano, Ana; Medina, Milagros

doi:10.5772/19305

Cited by 2 publications

References 40 publications

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Electron Transferases

Ferreira¹,

Martínez‐Júlvez²,

Medina³

2014

Methods in Molecular Biology

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The flavin isoalloxazine ring in electron transferases functions in a redox capacity, being able to take up electrons from a donor to subsequently deliver them to an acceptor. The main characteristics of these flavoproteins, including their unique ability to mediate obligatory processes of two-electron transfers with those involving single-electron transfer, are here described. To illustrate the versatility of these proteins, the acquired knowledge of the function of the two electron transferases involved in the cyanobacterial photosynthetic electron transfer from photosystem I to NADP(+) is presented. Many aspects of their biochemistry and biophysics have been extensively characterized using site-directed mutagenesis, steady-state and transient kinetics, spectroscopy, calorimetry, X-ray crystallography, electron paramagnetic resonance, and computational methods.

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Electron Transferases

Ferreira¹,

Martínez‐Júlvez²,

Medina³

2014

Methods in Molecular Biology

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Direct examination of the relevance for folding, binding and electron transfer of a conserved protein folding intermediate

Lamazares

Vega

Ferreira

et al. 2017

Phys. Chem. Chem. Phys.

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Near the minimum free energy basin of proteins where the native ensemble resides, partly unfolded conformations of slightly higher energy can be significantly populated under native conditions. It has been speculated that they play roles in molecular recognition and catalysis, but they might represent contemporary features of the evolutionary process without functional relevance. Obtaining conclusive evidence on these alternatives is difficult because it requires comparing the performance of a given protein when populating and when not populating one such intermediate, in otherwise identical conditions. Wild type apoflavodoxin populates under native conditions a partly unfolded conformation (10% of molecules) whose unstructured region includes the binding sites for the FMN cofactor and for redox partner proteins. We recently engineered a thermostable variant where the intermediate is no longer detectable. Using the wild type and variant, we assess the relevance of the intermediate comparing folding kinetics, cofactor binding kinetics, cofactor affinity, X-ray structure, intrinsic dynamics, redox potential of the apoflavodoxin-cofactor complex (Fld), its affinity for partner protein FNR, and electron transfer rate within the Fld/FNR physiological complex. Our data strongly suggest the intermediate state, conserved in long-chain apoflavodoxins, is not required for the correct assembly of flavodoxin nor does it contribute to shape its electron transfer properties. This analysis can be applied to evaluate other native basin intermediates.

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Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+-Reductase

Cited by 2 publications

References 40 publications

Electron Transferases

Electron Transferases

Direct examination of the relevance for folding, binding and electron transfer of a conserved protein folding intermediate

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