X-ray dose-dependent structural changes of the [2Fe-2S] ferredoxin from Chlamydomonas reinhardtii.

Plant-type ferredoxin (Fd) is an electron transfer protein in chloroplast. Redox-dependent structural change of Fd controls its association with and dissociation from Fd-dependent enzymes. Among many X-ray structures of oxidized Fd have been reported so far, very likely a given number of them was partially reduced by strong X-ray. To understand the precise structural change between reduced and oxidized Fd, it is important to know whether the crystals of oxidized Fd may or may not be reduced during the X-ray experiment. We prepared the thin plate-shaped Fd crystals from Chlamydomonas reinhardtii and monitored its absorption spectra during experiment. Absorption spectra of oxidized Fd crystals were clearly changed to that of reduced form in an X-ray dose-dependent manner. In another independent experiment, the X-ray diffraction images obtained from different parts of one single crystal were sorted and merged to form two datasets with low and high X-ray doses. An Fo-Fo map calculated from the two datasets showed that X-ray reduction causes a small displacement of the iron atoms in the [2Fe-2S] cluster. Both our spectroscopic and crystallographic studies confirm X-ray dose-dependent reduction of Fd, and suggest a structural basis for its initial reduction step especially in the core of the cluster.

Reproducible radiation-damage processes in proteins irradiated by intense x-ray pulses.

X-ray free-electron lasers have enabled femtosecond protein nanocrystallography, a novel method to determine the structure of proteins. It allows time-resolved imaging of nanocrystals that are too small for conventional crystallography. The short pulse duration helps in overcoming the detrimental effects of radiation damage because x rays are scattered before the sample has been significantly altered. It has been suggested that, fortuitously, the diffraction process self-terminates abruptly once radiation damage destroys the crystalline order. Our calculations show that high-intensity x-ray pulses indeed trigger a cascade of damage processes in ferredoxin crystals, a particular metalloprotein of interest. However, we found that the damage process is initially not completely random. Correlations exist among the protein monomers, so that Bragg diffraction still occurs in the damaged crystals, despite significant atomic displacements. Our results show that the damage process is reproducible to a certain degree, which is potentially beneficial for the orientation step in single-molecule imaging.

Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Laser flash-induced photoreduction of photosynthetic ferredoxins and flavodoxin by 5-deazariboflavin and by a viologen analogue.

Laser flash photolysis has been used to compare the kinetics of reduction of ferredoxin isoforms from the green alga Monoraphidium braunii, and the ferredoxin and flavodoxin from the cyanobacterium Anabaena PCC 7119, by 5-deazariboflavin semiquinone (dRfH.) and the viologen analogue 1,1'-propylene-2,2'-bipyridyl (PDQ.+). Similar ionic strength-independent second-order rate constants (1.4 x 10(8) M-1 s-1) were obtained for the reduction of both algal ferredoxin isoforms by dRfH.. For the reduction of oxidized flavodoxin by dRfH., a more complex behavior was observed, with a second-order rate constant for dRfH. decay of 1.8 x 10(8) M-1 s-1, and a first-order (i.e. protein concentration independent) rate constant of 450 s-1, that probably corresponds to the protonation of the FMN semiquinone cofactor, which occurs subsequent to electron transfer. A value of 5 x 10(7) M-1 s-1 was obtained for the second-order rate constant of flavodoxin semiquinone reduction by dRfH.. The reduction of ferredoxins and flavodoxin semiquinone by PDQ.+ showed nonlinear protein concentration dependencies, consistent with a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer. A negative ionic strength effect on the kinetic constants was obtained, indicating the existence of attractive electrostatic interactions during electron transfer. With all the ferredoxins the k infinity values (rate constants extrapolated to infinite ionic strength) for the second-order step of the reduction process (complex formation) are smaller than previously reported for spinach ferredoxin, although Anabaena ferredoxin is somewhat more reactive than are the algal ferredoxins with the viologen.(ABSTRACT TRUNCATED AT 250 WORDS)

The pea ferredoxin I gene exhibits different light responses in pea and tobacco.

We monitored Fed-1 (encoding ferredoxin I) mRNA levels in etiolated transgenic tobacco seedlings containing the intact pea Fed-1 gene to determine if the characteristic light responses of this gene in pea seedlings are also observed in transgenic tobacco. Fed-1 transcript levels in transgenic tobacco seedlings closely paralleled those of the native gene in pea buds when etiolated seedlings were transferred to white light. However, the response to red light was much smaller in tobacco than in pea and was not efficiently reversed by far-red light. The red light response of endogenous tobacco ferredoxin transcripts is closely comparable to that of the Fed-1 transgene, with a similar lack of photoreversibility. Thus, the pea Fed-1 transgene responds normally to tobacco gene-regulatory factors, but these factors are less influenced by phytochrome in tobacco cotyledons than in pea buds.

Primary reactions of photosynthesis: photoreduction of a bound chloroplast ferredoxin at low temperature as detected by EPR spectroscopy.

An electron paramagnetic resonance signal was observed at 25 degrees K in whole spinach chloroplasts after illumination at 77 degrees K. The light-induced epr spectrum had g-values (g(x) = 1.86, g(y) = 1.94, g(z) = 2.05) and a temperature dependence that were characteristic of the reduced state of a plant-type ferredoxin. The light-induced epr spectrum was also observed in broken spinach chloroplasts from which soluble ferredoxin was removed. Chemical analyses showed that both whole and broken spinach chloroplasts contained amounts of nonheme iron and "acid-labile sulfide" consistent with the presence of a bound iron-sulfur protein, at a level of about one molecule per 75 chlorophyll molecules. These results support the conclusion that chloroplasts contain a bound ferredoxin that may serve as a primary low-potential electron acceptor in photosynthesis.