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

Abstract

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium <i>Thermochromatium tepidum</i> requires Ca²⁺ 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 <i>Tch. tepidum</i> LH1 complex in an engineered <i>Rhodobacter sphaeroides</i> mutant strain. This system contained a chimeric <i>pufBALM</i> gene cluster (<i>pufBA</i> from <i>Tch. tepidum</i> and <i>pufLM</i> from <i>Rba. sphaeroides</i>) 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 <i>Tch. tepidum</i> 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 <i>Rba. sphaeroides</i>) was incorporated into the <i>Tch. tepidum</i> LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in <i>Rba. sphaeroides</i> 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 <i>Tch. tepidum</i> LH1 complex; in contrast, α-N50 does not participate in Ca²⁺ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated <i>Tch. tepidum</i> LH1-RC complex and have unambiguously identified the location of Ca²⁺ within this key antenna complex.