Probing structure-function relationships in early events in photosynthesis using a chimeric photocomplex.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium requires Ca for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the LH1 complex in an engineered mutant strain. This system contained a chimeric gene cluster ( from and from ) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in ) was incorporated into the LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the LH1 complex; in contrast, α-N50 does not participate in Ca coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated LH1-RC complex and have unambiguously identified the location of Ca within this key antenna complex.