Volkmar H. R. Schmid

Requirement for the H Phosphoprotein in Photosystem II of Chlamydomonas reinhardtii

To dissect the expression of the psbB gene cluster of the Chlamydomonas reinhardtii chloroplast genome and to assess the role of the photosystem II H-phosphoprotein (PSII-H) in the biogenesis and/or stabilization of PSII, an aadA gene cassette conferring spectinomycin resistance was employed for mutagenesis. Disruption of the gene cluster has no effect on the abundance of transcripts of the upstream psbB/T locus. Likewise, interruption of psbB/T and psbH with a strong transcriptional terminator from the rbcL gene does not influence transcript accumulation. Thus, psbB/T and psbH may be independently transcribed, and the latter gene seems to have its own promoter in C. reinhardtii. In the absence of PSII-H, translation and thylakoid insertion of chloroplast PSII core proteins is unaffected, but PSII proteins do not accumulate. Because the deletion mutant also exhibits PSII deficiency when dark-grown, the effect is unrelated to photoinhibition. Turnover of proteins B and C of PSII and the polypeptides PSII protein A and PSII protein D is faster than in wild-type cells but is much slower than that observed in other PSII-deficient mutants of C. reinhardtii, suggesting a peripheral location of PSII-H in PSII. The role of PSII-H on PSII assembly was examined by sucrose gradient fractionation of pulse-labeled thylakoids; the accumulation of high-molecular-weight forms of PSII is severely impaired in the psbH deletion mutant. Thus, a primary role of PSII-H may be to facilitate PSII assembly/stability through dimerization. PSII-H phosphorylation, which possibly occurs at two sites, may also be germane to its role in regulating PSII structure, stabilization, or activity.

Pigment Binding of Photosystem I Light-harvesting Proteins

Light-harvesting complexes (LHC) of higher plants are composed of at least 10 different proteins. Despite their pronounced amino acid sequence homology, the LHC of photosystem II show differences in pigment binding that are interpreted in terms of partly different functions. By contrast, there is only scarce knowledge about the pigment composition of LHC of photosystem I, and consequently no concept of potentially different functions of the various LHCI exists. For better insight into this issue, we isolated native LHCI-730 and LHCI-680. Pigment analyses revealed that LHCI-730 binds more chlorophyll and violaxanthin than LHCI-680. For the first time all LHCI complexes are now available in their recombinant form; their analysis allowed further dissection of pigment binding by individual LHCI proteins and analysis of pigment requirements for LHCI formation. By these different approaches a correlation between the requirement of a single chlorophyll species for LHC formation and the chlorophylla/b ratio of LHCs could be detected, and indications regarding occupation of carotenoid-binding sites were obtained. Additionally the reconstitution approach allowed assignment of spectral features observed in native LHCI-680 to its components Lhca2 and Lhca3. It is suggested that excitation energy migrates from chlorophyll(s) fluorescing at 680 (Lhca3) via those fluorescing at 686/702 nm (Lhca2) or 720 nm (Lhca3) to the photosystem I core chlorophylls.

Lhca5 interaction with plant photosystem I.

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light‐harvesting protein of photosystem I (Lhca)‐type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross‐linking studies on PSI‐LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light‐harvesting protein with LHCI in the Lhca2/Lhca3‐site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding‐competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light‐harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b‐antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl‐binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77‐K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long‐wavelength absorption and 730‐nm fluorescence emission. In Lhcb1 with only Chl b long‐wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long‐wavelength properties.

Ultrafast excitation dynamics of low energy pigments in reconstituted peripheral light-harvesting complexes of photosystem I

Ultrafast dynamics of a reconstituted Lhca4 subunit from the peripheral LHCI‐730 antenna of photosystem I of higher plants were probed by femtosecond absorption spectroscopy at 77 K. Intramonomeric energy transfer from chlorophyll (Chl) b to Chl a and energy equilibration between Chl a molecules observed on the subpicosecond time scale are largely similar to subpicosecond energy equilibration processes within LHCII monomers. However, a 5 ps equilibration process in Lhca4 involves unique low energy Chls in LHCI absorbing at 705 nm. These pigments localize the excitation both in the Lhca4 subunit and in LHCI‐730 heterodimers. An additional 30–50 ps equilibration process involving red pigments of Lhca4 in the heterodimer, observed by transient absorption and picosecond fluorescence spectroscopy, was ascribed to intersubunit energy transfer.

Protein domains required for formation of stable monomeric Lhca1- and Lhca4-complexes

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHC I-680 and LHC I-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHC I using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHC I, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHC I versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHC II and CP29.

Molecular origin of red pigments in a peripheral light-harvesting antenna of Photosystem I: ultrafast absorption spectroscopy of recombinant Lhca4

A remarkable feature of Photosystem I in higher plants and green algae are chlorophylls absorbing and emitting at energies lower than P700. A dominant portion of the red fluorescence comes from a peripheral light-harvesting antenna (LHCI), specifically from its LHCI-730 subpopulation. One of the constituents of the LHCI730 heterodimer, the Lhca4 subunit, was found to harbor the low energy (red) pigments emitting at 730 nm (Tjus et al. 1995; Schmid et al. 1997; Knoetzel et al. 1998). Excitation energy transfer processes in the LHCI-730 and molecular organization of the red pigments that lead to the excitation energy localization are poorly understood. 77 K transient absorption difference spectra of Lhca4 upon excitation of Chl b revealed the presence of several ultrafast energy transfer processes with yet-unresolved lifetimes (Melkozernov et al. 2000a). Two major energy transfer processes include a 400-600 fs energy transfer between spectral forms of Chl b and Chl a followed by 3-5 ps energy equilibration between Chl a molecules including those involved in the red pigment’s transition at 705 nm. A significant red shift of the C-705 spectral form as compared to the major Chl a spectral form at 679 nm might be a result of a strong excitonic interaction of the Chl a molecules localizing excitations in the Lhca4. Ihalainen et al. (2000) suggested the presence of one Chl a dimer per LHCI-730 based on steady state spectroscopy of native LHCI complexes. Recently, Schmid et al. (2001) obtained evidence of Chl b involvement in the long-wavelength transition in Lhca4 based on steady state spectroscopy analysis of reconstituted Lhca4. In this work we use ultrafast transient absorption spectroscopy at 8 K to probe coupling of Chl a and Chl b in the recombinant Lhca4 polypeptides with changed pigment occupancy as compared to the wild type Lhca4.