A water-soluble yellow protein, previously discovered in the purple photosynthetic bacterium Ectothiorhodospira halophila, contains a chromophore which has an absorbance maximum at 446 nm. The protein is now shown to be photoactive. A pulse of 445-nm laser light caused the 446-nm peak to be partially bleached and red-shifted in a time less than 1 microsecond. The intermediate thus formed was subsequently further bleached in the dark in a biphasic process occurring in approximately 20 ms. Finally, the absorbance of native protein was restored in a first-order process occurring over several seconds. These kinetic processes are remarkably similar to those of sensory rhodopsin from Halobacterium, and to a lesser extent bacteriorhodopsin and halorhodopsin; although these proteins are membrane-bound, they have absorbance maxima at about 570 nm, and they cycle more rapidly. In attempts to remove the chromophore for identification, it was found that a variety of methods of denaturation of the protein caused transient or permanent conversion to a form which has an absorbance maximum near 340 nm. Thus, by analogy to the rhodopsins, the absorption at 446 nm in the native protein appears to result from a 106-nm red shift of the chromophore induced by the protein. Acid denaturation followed by extraction with organic solvents established that the chromophore could be removed from the protein. It is not identical with all-trans-retinal and remains to be identified, although it could still be a related pigment. The E. halophila yellow protein has a circular dichroism spectrum which indicates little alpha-helical secondary structure (19%).(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in

To understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP). Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic level. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein. Glu46, replaced by Gln, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state. The Glu46Gln mutation shifted the ground state absorption maximum from 446 to 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds. Arg52, which separates the active site from solvent in the ground state, was substituted by Ala. The smaller red shift (to 452 nm) of the Arg52Ala mutant suggests that electrostatic interactions with Arg52 are not important for charge stabilization on the chromophore. Both mutations cause interesting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu46Gln PYP in response to a change in pH from pH 5 to 10 (pKa = 8). Prompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle. The rate of color loss decreased about 3-fold with increasing pH from pH 5 to 10. The rate of recovery to the colored ground state showed a bell-shaped pH dependence, controlled by two pKa values (6.4 and 9.4). The maximum recovery rate at pH 7.9 is about 16 times faster than at pH 5. The effect of pH on Arg52Ala is like that on wild type except for faster loss of color and slower recovery. These kinetic effects of the mutations and the changes with pH demonstrate that both phases in PYP's light cycle are actively controlled by the protein component.

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-Å resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle ‫؍‬ 170°) and the third (type II) copper lies only 3.1 Å from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 M Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k ‫؍‬ 4 ؋ 10 7 M ؊1 ⅐s ؊1 ), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k ‫؍‬ 9 ؋ 10 7 M ؊1 ⅐s ؊1 , followed by a slow intramolecular electron transfer to T1 copper (k ϳ10 s ؊1 ). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.