Modification of two peptides of bacteriorhodopsin with a (pentaammine)cobalt(III) complex

Abstract

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.

Location of a Cation-Binding Site in the Loop between Helices F and G of Bacteriorhodopsin as Studied by 13C NMR

“…A study of emission lifetimes of bound Eu 3ϩ in the Eu 3ϩ -regenerated purple membrane has also shown equivalent binding constants for Eu 3ϩ ions corresponding to three observed decay components of emission (Corcoran et al, 1987). The rapid exchange of the cations observed in this study seems to be consistent with the finding that the cation-binding sites are located on the surface (Mitra and Stroud, 1990;Fu et al, 1997;Engelhard et al, 1989), even though the changes in the peaks at 15.88 ppm and 16.40 ppm for the Mn 2ϩ and Na ϩ purple membrane show that parts of the ␣ II -helices are close to the cation-binding sites. Thus, although the findings in this study do not rule out the possibility of cation-binding sites in the transmembrane region of bR, we expect from the NMR results that the cation-binding sites are near the hydrophilic surface and thus are easily accessible to the medium.…”

D38 Is an Essential Part of the Proton Translocation Pathway in Bacteriorhodopsin

“…Therefore, changes in the kinetics of the photocycle and the pumping cycle can solely be attributed to functional differences in the exchanged amino acids. Incubation of cation-depleted BR with Fe 3+ and pentaamineaquocobalt(III) tetrafluoroborate led to a drastic retardation of the M decay (Engelhard et al, 1989(Engelhard et al, , 1990. The data suggest that carboxyl groups on the CP surface of BR are involved in the interaction with these compounds, although the definite site of modification/interaction is unknown.…”

PURPLE MEMBRANE: SURFACE CHARGE DENSITY and THE MULTIPLE EFFECT OF pH and CATIONS

“…Modification of bR carboxyl groups by the chemical addition of [Co(NH&I3+ has been investigated, and a stable complex was observed attached to sequences on the C-terminal side of the membrane, but only after the membrane was illuminated (Engelhard et al, 1989). A further investigation of the location of a cation bound in this region is currently under investigation (Engelhard et al, 1990;abstract).…”