Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880 nm to 915 nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes

Biohybrid Photosynthetic Antenna Complexes for Enhanced Light-Harvesting

Biohybrid antenna systems have been constructed that contain synthetic chromophores attached to 31mer analogues of the bacterial photosynthetic core light-harvesting (LH1) β-polypeptide. The peptides are engineered with a Cys site for bioconjugation with maleimide-terminated chromophores, which include synthetic bacteriochlorins (<b>BC1</b>, <b>BC2</b>) with strong near-infrared absorption and commercial dyes Oregon green (<b>OGR</b>) and rhodamine red (<b>RR</b>) with strong absorption in the blue-green to yellow-orange regions. The peptides place the Cys 14 (or 6) residues before a native His site that binds bacteriochlorophyll <i>a</i> (BChl-<i>a</i>) and, like the native LH proteins, have high helical content as probed by single-reflection IR spectroscopy. The His residue associates with BChl-<i>a</i> as in the native LH1 β-polypeptide to form dimeric ββ-subunit complexes [31mer­(−14Cys)­X/BChl]₂, where X is one of the synthetic chromophores. The native-like BChl-<i>a</i> dimer has Q_<i>y</i> absorption at 820 nm and serves as the acceptor for energy from light absorbed by the appended synthetic chromophore. The energy-transfer characteristics of biohybrid complexes have been characterized by steady-state and time-resolved fluorescence and absorption measurements. The quantum yields of energy transfer from a synthetic chromophore located 14 residues from the BChl-coordinating His site are as follows: <b>OGR</b> (0.30) < <b>RR</b> (0.60) < <b>BC2</b> (0.90). Oligomeric assemblies of the subunit complexes [31mer­(−14Cys)­X/BChl]_<i>n</i> are accompanied by a bathochromic shift of the Q_<i>y</i> absorption of the BChl-<i>a</i> oligomer as far as the 850-nm position found in cyclic native photosynthetic LH2 complexes. Room-temperature stabilized oligomeric biohybrids have energy-transfer quantum yields comparable to those of the dimeric subunit complexes as follows: <b>OGR</b> (0.20) < <b>RR</b> (0.80) < <b>BC1</b> (0.90). Thus, the new biohybrid antennas retain the energy-transfer and self-assembly characteristics of the native antenna complexes, offer enhanced coverage of the solar spectrum, and illustrate a versatile paradigm for the construction of artificial LH systems