Identification of a single peridinin sensing Chl-a excitation in reconstituted PCP by crystallography and spectroscopy

Abstract

The peridinin-chlorophyll a-protein (PCP) of dinoflagellates is unique among the large variety of natural photosynthetic light-harvesting systems. In contrast to other chlorophyll protein complexes, the soluble PCP is located in the thylakoid lumen, and the carotenoid pigments outnumber the chlorophylls. The structure of the PCP complex consists of two symmetric domains, each with a central chlorophyll a (Chl-a) surrounded by four peridinin molecules. The protein provides distinctive surroundings for the pigment molecules, and in PCP, the specific environment around each peridinin results in overlapping spectral line shapes, suggestive of different functions within the protein. One particular Per, Per-614, is hypothesized to show the strongest electronic interaction with the central Chl-a. We have performed an in vitro reconstitution of pigments into recombinant PCP apo-protein (RFPCP) and into a mutated protein with an altered environment near Per-614. Steady-state and transient optical spectroscopic experiments comparing the RFPCP complex with the reconstituted mutant protein identify specific amino acid-induced spectral shifts. The spectroscopic assignments are reinforced by a determination of the structures of both RFPCP and the mutant by x-ray crystallography to a resolution better than 1.5 A. RFPCP and mutated RFPCP are unique in representing crystal structures of in vitro reconstituted light-harvesting pigment-protein complexes.

Fig. 1.

Structure of RFPCP. Two protein subunits (black and gray) form a dimer enclosing the pigment molecules (Chl-a in green and Per in orange). N- and C-terminus and Per numbers of the subunits are labeled.

Fig. 2.

Superimposition of the protein helices of RFPCP (gray) and MFPCP (yellow) and the pigments of RFPCP (colored) and MFPCP (black). N- and C-terminus of the MFPCP protein, as well as MFPCP Per numbers, are labeled.

Fig. 3.

Binding site of Per-614 in (A) RFPCP and (B) N89L mutant. Per-614, Chl-601, and the mutated residue are shown as thick lines; other Pers are shown as thin lines. Omit electron-density maps are shown for Per-614 and the mutated residue in RFPCP (contoured at 5σ) and the N89L mutant (4σ).

Fig. 4.

(A) Absorption spectra of RFPCP and N89L mutant taken at 10 K. Both spectra were normalized at Qy band. The N89L-RFPCP absorption difference steady-state spectrum (10 K) indicates the most prominent spectral shifts. (B) Reconstruction of the RFPCP and N89L 10-K absorption spectra. RFPCP and N89L are fitted with the same parameters except for Per-4, which is shifted by ≈24 nm and decreased in intensity by ≈8% in the N89L mutant. See text and Origin of the absorption shift in the N89L 10-K absorption spectrum in the SI Text for further details.

Fig. 5.

TA spectra of RFPCP and N89L measured at 10 K. The samples were excited in the Per absorption region at 475 nm and into the Qy band of Chl-a at 670 nm.

Fig. 6.

Overlay of the 10-K TA spectra of N89L and RFPCP taken 5 ps after Chl-a Qy excitation.

Fig. 7.

Calculation of the second derivatives of the full 10-K absorption spectra of RFPCP and N89L as well as of the 10-K Per-614 spectra of RFPCP and N89L. The calculated spectra are overlaid with the dip from TA measurements measured 50 ps after Chl-a excitation. The 10-K absorption spectra of RFPCP and N89L are included for better identification of the spectral region.