bilin ͉ biliprotein ͉ photochromic ͉ histidine kinase

The P r 3 P fr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R C were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in P r and ZZEssa in P fr , with all pyrrole nitrogens being protonated. Photoexcitation of P r is followed by (at least) three thermal relaxation components in the formation of P fr with time constants of 230 s and 3.1 and 260 ms. H 2 O/D 2 O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R C , respectively. Resonance Raman measurements of Meta-R C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P r 3 P fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the P fr formation that switch between the active and the inactive state of the photoreceptor.Phytochromes are photoreceptors that utilize light as a source of information for controlling numerous biological processes (1, 2). The chromophore, a methine-bridged tetrapyrrole ( Fig. 1), acts as a photoswitch between two stable, spectrally distinct forms, denoted as P r and P fr according to the red and far-red absorption maxima, respectively. The P r /P fr interconversion is initiated by the rapid Z/E photoisomerization of the C-D methine bridge (3), followed by chromophore relaxations that are coupled to structural changes of the apoprotein (4). These structural changes are the trigger for signal transduction. Resonance Raman (RR) 2 and IR spectroscopy have provided valuable insight into light-induced chromophore and protein structural changes (e.g. see Refs. 5-10), but molecular and mechanistic details are not yet known and no crystal structure of a phytochrome has been reported so far.While phytochromes were originally thought to be restricted to plants, the discovery of these chromoproteins in cyanobacteria (11) and other bacteria points to the prokaryotic origin of this family of photoreceptors. In contrast to plant phytochromes, typical bacterial phytochromes are light-regulated histidine kinases. Despite the quite different regulatory functions (12, 13), plant and bacterial phytochromes exhibit structural and mechanistic similarities. The phytochromobilin chromophore of plant phytoc...

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The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK a from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc 3 Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

following correction should be noted. Due to an editorial change at PNAS, the meaning of the last sentence on page 14046 was altered. The sentence originally read as follows: On the other hand, this structure does not reproduce the pharmacological properties of either P or Q channel exactly, as the ID 50 to sFTX and -Aga IVA for P-type channels is lower than for the ␣1A, ␣2␦, ␤Ib channels in HEK cells.Neurobiology. In the article "The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide" Kuo Wu, Chiye Aoki, Alice Elste, Adrienne A. Rogalski-Wilk, and Philip Siekevitz, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(13273)(13274)(13275)(13276)(13277)(13278), the quality of the reproduction of Fig. 2A was poor. The figure and its legend are shown below:Biochemistry. In the article "KSR stimulates Raf-1 activity in a kinase-independent manner" by Neil R. Michaud, Marc Therrien, Angela Cacace, Lisa C. Edsall, Sarah Spiegel, Gerald M. Rubin, and Deborah K. Morrison, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(12792)(12793)(12794)(12795)(12796), the following correction should be noted.Due to a printer's error, background was incorrectly added to (50 g) and 100 g each of the other fractions, in 100 l final volume, including whole homogenate (H), synaptosomes (Syn), synaptic plasma membranes (SPM), and crude synaptic vesicles (CSV), were incubated at 37°C for 15 min. NAD incorporation was performed in the absence (Ϫ) or presence (ϩ) of SNP as exogenous source of NO. The mixtures were subjected to SDS͞PAGE and then autoradiography. (B) Western blot analysis of the G3PD in the subcellular fractions. To confirm that the radioactive protein in the subcellular fractions was indeed G3PD, Western blot analysis was performed by using specific anti-G3PD antibodies as described.

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.

The P(r) to P(fr) transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and its N-terminal sensor domain Cph1Delta2 is accompanied by net acidification in unbuffered solution. The extent of this net photoreversible proton release was measured with a conventional pH electrode and increased from less than 0.1 proton released per P(fr) formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph1Delta2) H(+)/P(fr) at pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transient absorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constants of approximately 4 and approximately 20 ms that were also observed in parallel measurements of the photocycle (tau(3) and tau(4)). The number of transiently released protons per P(fr) formed is about one. This H(+) release phase is followed by a proton uptake phase of a smaller amplitude that has a time constant of approximately 270 ms (tau(5)) and is synchronous with the formation of P(fr). The acidification observed in the P(r) to P(fr) transition with pH electrodes is the net effect of these two sequential protonation changes. Flash-induced transient absorption measurements were carried out with Cph1 and Cph1Delta2 at pH 7 and pH 8. Global analysis indicated the presence of five kinetic components (tau(1)-tau(5): 5 and 300 micros and 3, 30, and 300 ms). Whereas the time constants were approximately pH independent, the corresponding amplitude spectra (B(1), B(3), and B(5)) showed significant pH dependence. Measurements of the P(r)/P(fr) photoequilibrium indicated that it is pH independent in the range of 6.5-9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested that the phycocyanobilin chromophore deprotonates at alkaline pH in both P(r) and P(fr) with an approximate pK(a) of 9.5. The protonation state of the chromophore at neutral pH is therefore the same in both P(r) and P(fr). The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pK(a) changes in the protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms after photoexcitation. These transient structural changes may be relevant for signal transduction by this cyanobacterial phytochrome.

The (6-4) photolyases use blue light to reverse UV-induced (6-4) photoproducts in DNA. This (6-4) photorepair was thought to be restricted to eukaryotes. Here we report a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens, and propose that (6-4) photolyases are broadly distributed in prokaryotes. The crystal structure of photolyase related protein B (PhrB) at 1.45 Å resolution suggests a DNA binding mode different from that of the eukaryotic counterparts. A His-His-X-X-Arg motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. The PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore and a [4Fe-4S] cluster bound to the catalytic domain. A significant part of the Fe-S fold strikingly resembles that of the large subunit of eukaryotic and archaeal primases, suggesting that the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. Our study presents a unique prokaryotic (6-4) photolyase and proposes that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family.DNA repair | UV light | energy transfer