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Impact of the N5-proximal Asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase.

ABSTRACT: Flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. A versatile regulatory tool is proton transfer. Here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (PLs) and cryptochromes (CRYs). These light-responsive proteins share homologous overall architectures and FAD-binding pockets, yet they have evolved divergent functions that include DNA repair, photomorphogenesis, regulation of circadian rhythm, and magnetoreception. We report the first measurement of both FAD redox potentials for cyclobutane pyrimidine dimer PL (CPD-PL, Anacystis nidulans). These values, E(1)(hq/sq) = -140 mV and E(2)(sq/ox) = -219 mV, where hq is FAD hydroquinone and ox is oxidized FAD, establish that the sq is not thermodynamically stabilized (?E = E(2) - E(1) = -79 mV). Results with N386D CPD-PL support our earlier hypothesis of a kinetic barrier to sq oxidation associated with proton transfer. Both E(1) and E(2) are upshifted by ? 100 mV in this mutant; replacing the N5-proximal Asn with Asp decreases the driving force for sq oxidation. However, this Asp alleviates the kinetic barrier, presumably by acting as a proton shuttle, because the sq in N386D CPD-PL oxidizes orders of magnitude more rapidly than wild type. These data clearly reveal, as suggested for plant CRYs, that an N5-proximal Asp can switch on proton transfer and modulate sq reactivity. However, the effect is context-dependent. More generally, we propose that PLs and CRYs tune the properties of their N5-proximal residue to adjust the extent of proton transfer, H-bonding patterns, and changes in protein conformation associated with electron transfer at the flavin.

SUBMITTER: Damiani MJ

PROVIDER: S-EPMC3039355 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Impact of the N5-proximal Asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase.

Damiani Michael J MJ Nostedt Jordan J JJ O'Neill Melanie A MA

The Journal of biological chemistry 20101203 6

Flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. A versatile regulatory tool is proton transfer. Here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (PLs) and cryptochromes (CRYs). These light-responsive proteins share homologous overall architectures and FAD-binding pockets, yet they have evolved divergent functions that include DNA repair, phot ...[more]

PMID: 21131361

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Project description:BackgroundMolecular phenomena driving pathological aggregation in neurodegenerative diseases are not completely understood yet. Peculiar is the case of Spinocerebellar Ataxia 3 (SCA3) where the conformational properties of the AT-3 N-terminal region, also called Josephin Domain (JD), play a key role in the first step of aggregation, having the JD an amyloidogenic propensity itself. For this reason, unraveling the intimate relationship between JD structural features and aggregation tendency may lead to a step forward in understanding the pathology and rationally design a cure. In this connection, computational modeling has demonstrated to be helpful in exploring the protein molecular dynamics and mechanism of action.ResultsConformational dynamics of the JD is here finely investigated by replica exchange molecular dynamics simulations able to sample the microsecond time scale and to provide both a thermodynamic and kinetic description of the protein conformational changes. Accessible structural conformations of the JD have been identified in: open, intermediate and closed like arrangement. Data indicated the closed JD arrangement as the most likely protein arrangement. The protein transition from closed toward intermediate/open states was characterized by a rate constant higher than 700 ns. This result also explains the inability of classical molecular dynamics to explore transitions from closed to open JD configuration on a time scale of hundreds of nanoseconds.ConclusionThis work provides the first kinetic estimation of the JD transition pathway from open-like to closed-like arrangement and vice-versa, indicating the closed-like arrangement as the most likely configuration for a JD in water environment. More widely, the importance of our results is also underscored considering that the ability to provide a kinetic description of the protein conformational changes is a scientific challenge for both experimental and theoretical approaches to date.ReviewersThis article was reviewed by Oliviero Carugo, Bojan Zagrovic.

Dataset Information

Impact of the N5-proximal Asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase.

Publications

Impact of the N5-proximal Asn on the thermodynamic and kinetic stability of the semiquinone radical in photolyase.

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