The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)

The phototaxis of Halobacterium halobium is initiated by two photoreceptors, the sensory rhodopsins sR-I and sR-11. An sR-11-like pigment has also been described in Natronobacterium pharaonis. In this work it was shown that N. pharaonis cells are repelled by light with a wavelength of 500 nm. A further comparison of membrane preparations from H . halobium (mutant DI) containing only sR-I1 and from N . pharaonis [strain SP1 (28)] with a chromophoric protein (psR-11) resembling sR-I1 revealed substantial similarities. The biochemical and photochemical properties of the pigments are quite similar, with psR-I1 being more stable to external conditions such as pH and ionic strength of the buffer. Both pigments are bleached by low concentrations of hydroxylamine and can be reconstituted by the addition of all-trans-retinal. The absorption spectrum of psR-I1 is quite similar to sR-I1 including the shoulder on the short-wavelength side. After light excitation sR-I1 and psR-I1 undergo photocycles with at least three intermediates. The earliest intermediate has an absorption maximum above 520 nm and decays to a species which has a characteristic absorption (z 380 nm) of a deprotonated Schiff base. The final step is the regeneration of the original ground state via a red-shifted intermediate absorbing around 540 nm. From this cumulative evidence it can be concluded that, not only sR-11, but also the pigment from N . pharaonis is a photophobic photoreceptor.

Bacteriorhodopsin (bR) was regenerated from the cation-depleted blue membrane with pentaammineaquocobalt(III) tetrafluoroborate [( Co(NH3)5H2O]3+[BF4-]3). Illumination of the sample with orange light decreased the extinction at 568 nm concomitantly with a hypsochromic shift of the absorption maximum. The photocycle of this sample was inhibited, and the rate of proton pumping was reduced. Chymotryptic cleavage of the corresponding apomembrane into the two fragments C1 and C2 and their subsequent separation revealed that cobalt label is only attached to C1. The maximal incorporation of Co into this peptide was 0.3 Co/C1. After cleavage of C1 with cyanogen bromide and subsequent proteolysis with trypsin and chymotrypsin, this modification could be associated with peptides from cyanogen bromide fragments 6 and 9. The sequences were determined to be 101Val-Asp-Ala-Asp-Gln and 228Ala-Ile-Phe-Gly-Glu-Ala-Glu-Ala. These peptides contain the sequences Asp-Ala-Asp and Glu-Ala-Glu, respectively, which might be constituents of the same cation binding site. The observation that the incorporation of Co into bacteriorhodopsin is enhanced under illumination with orange light indicates that this site might be involved in the proton uptake.