Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus.

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

Interaction of the Full-Length Heme-Based CO Sensor Protein RcoM-2 with Ligands.

The heme-based and CO-responsive RcoM transcriptional regulators from Burkholderia xenovorans are known to display an extremely high affinity for CO while being insensitive to O2. We have quantitatively characterized the heme-CO interaction in full-length RcoM-2 and compared it with the isolated heme domain RcoMH-2 to establish the origin of these characteristics. Whereas the CO binding rates are similar to those of other heme-based sensor proteins, the dissociation rates are two to three orders of magnitude lower. The latter property is tuned by the yield of CO escape from the heme pocket after disruption of the heme-CO bond, as determined by ultrafast spectroscopy. For the full-length protein this yield is ∼0.5%, and for the isolated heme domain it is even lower, associated with correspondingly faster CO rebinding kinetics, leading to Kd values of 4 and 0.25 nM, respectively. These differences imply that the presence of the DNA-binding domain influences the ligand-binding properties of the heme domain, thus abolishing the observed quasi-irreversibility of CO binding to the isolated heme domain. RcoM-2 binds target DNA with high affinity (Kd < 2 nM) when CO is bound to the heme, and the presence of DNA also influences the heme-CO rebinding kinetics. The functional implications of our findings are discussed.

Fluorescent iron­sulfur centers: Photochemistry of the PetA Rieske protein from Aquifex aeolicus.

Cytochrome bc1 complexes are energy-transducing enzymes and key components of respiratory electron chains. They contain Rieske 2Fe2S proteins that absorb very weakly in the visible absorption region compared to the heme cofactors of the cytochromes, but are known to yield photoproducts. Here, the photoreactions of isolated Rieske proteins from the hyperthermophilic bacterium Aquifex aeolicus are studied in two redox states using ultrafast transient fluorescence and absorption spectroscopy. We provide evidence, for the first time in iron­sulfur proteins, of very weak fluorescence of the excited state, in the oxidized as well as the reduced state. The excited states of the oxidized and reduced forms decay in 1.5 ps and 30 ps, respectively. In both cases they give rise to product states with lifetimes beyond 1 ns, reflecting photo-reduction of oxidized centers as well as photo-oxidation of reduced centers. Potential reaction partners are discussed and studied using site-directed mutagenesis. For the reduced state, a nearby disulfide bridge is suggested as an electron acceptor. The resulting photoproducts in either state may play a role in photoactivation processes.

Regulation of the ROS response dynamics and organization to PDGF motile stimuli revealed by single nanoparticle imaging.

Although reactive oxygen species (ROS) are better known for their harmful effects, more recently, H2O2, one of the ROS, was also found to act as a secondary messenger. However, details of spatiotemporal organization of specific signaling pathways that H2O2 is involved in are currently missing. Here, we use single nanoparticle imaging to measure the local H2O2 concentration and reveal regulation of the ROS response dynamics and organization to platelet-derived growth factor (PDGF) signaling. We demonstrate that H2O2 production is controlled by PDGFR kinase activity and EGFR transactivation, requires a persistent stimulation, and is regulated by membrane receptor diffusion. This temporal filtering is impaired in cancer cells, which may determine their pathological migration. H2O2 subcellular mapping reveals that an external PDGF gradient induces an amplification-free asymmetric H2O2 concentration profile. These results support a general model for the control of signal transduction based only on membrane receptor diffusion and second messenger degradation.

Multifunctional rare-Earth vanadate nanoparticles: luminescent labels, oxidant sensors, and MRI contrast agents.

Collecting information on multiple pathophysiological parameters is essential for understanding complex pathologies, especially given the large interindividual variability. We report here multifunctional nanoparticles which are luminescent probes, oxidant sensors, and contrast agents in magnetic resonance imaging (MRI). Eu(3+) ions in an yttrium vanadate matrix have been demonstrated to emit strong, nonblinking, and stable luminescence. Time- and space-resolved optical oxidant detection is feasible after reversible photoreduction of Eu(3+) to Eu(2+) and reoxidation by oxidants, such as H2O2, leading to a modulation of the luminescence emission. The incorporation of paramagnetic Gd(3+) confers in addition proton relaxation enhancing properties to the system. We synthesized and characterized nanoparticles of either 5 or 30 nm diameter with compositions of GdVO4 and Gd0.6Eu0.4VO4. These particles retain the luminescence and oxidant detection properties of YVO4:Eu. Moreover, the proton relaxivity of GdVO4 and Gd0.6Eu0.4VO4 nanoparticles of 5 nm diameter is higher than that of the commercial Gd(3+) chelate compound Dotarem at 20 MHz. Nuclear magnetic resonance dispersion spectroscopy showed a relaxivity increase above 10 MHz. Complexometric titration indicated that rare-earth leaching is negligible. The 5 nm nanoparticles injected in mice were observed with MRI to concentrate in the liver and the bladder after 30 min. Thus, these multifunctional rare-earth vanadate nanoparticles pave the way for simultaneous optical and magnetic resonance detection, in particular, for in vivo localization evolution and reactive oxygen species detection in a broad range of physiological and pathophysiological conditions.

Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells.

Low concentrations of reactive oxygen species, notably hydrogen peroxide (H(2)O(2)), mediate various signalling processes in the cell. Production of these signals is highly regulated and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H(2)O(2) generation in living cells. The Y(0.6)Eu(0.4)VO(4) nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H(2)O(2) with a 30-s temporal resolution and a dynamic range of 1-45 microM. The differences in the timing of intracellular H(2)O(2) production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H(2)O(2), in many signalling processes H(2)O(2) is, however, the dominant oxidant. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species.