James A. Bautista

On the photophysics and photochemical properties of carotenoids and their role as light-harvesting pigments in photosynthesis

In photosynthetic organisms, carotenoids have been implicated in several diverse roles. Yet, owing to profound technical difficulties encountered in attempting to examine the electronic state energies and dynamics of carotenoids both in vitro and in vivo, several questions remain, and much of the data and interpretations of the results are controversial. This paper will discuss some of these questions and controversies, the resolution of which is important in unraveling the manner in which carotenoids function in photosynthetic systems.

The spectroscopic and photochemical properties of locked-15,15′-cis-spheroidene in solution and incorporated into the reaction center of Rhodobacter sphaeroides R-26.1

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15′-cis-spheroidene, were studied by absorption, fluorescence, circular dichroism, fast transient absorption and electron spin resonance spectroscopies in solution and after incorporation into the reaction center of Rhodobacter (Rb.) sphaeroides R-26.1. HPLC purification of the synthetic molecule reveals the presence of several di-cis geometric isomers in addition to the mono-cis isomer of locked-15,15′-cis-spheroidene. In solution, the absorption spectrum of the purified mono-cis sample was red-shifted and showed a large cis-peak at 351 nm compared to unlocked all-trans spheroidene. Molecular modeling and semi-empirical calculations reveal how geometric isomerization and structural factors affect the room temperature spectra. The spectroscopic studies of the purified locked-15,15′-mono-cis molecule in solution reveal a more stable manifold of excited states compared to the unlocked spheroidene. Reaction centers of Rb. sphaeroides R-26.1 in which the locked-15,15′-cis-spheroidene was incorporated show no difference in either the spectroscopic properties or photochemistry compared to reaction centers in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 reaction centers which naturally contain spheroidene. The data suggest that the natural selection of a cis-isomer of spheroidene for incorporation into native reaction centers of Rb. sphaeroides wild type strain 2.4.1 is more determined by the structure or assembly of the reaction center protein than by any special quality of the cis-isomer of the carotenoid that would affect its ability to participate in triplet energy transfer or carry out photoprotection.

Construction and Characterization of Genetically Modified Synechocystis sp. PCC 6803 Photosystem II Core Complexes Containing Carotenoids with Shorter π-Conjugation than β-Carotene

β-Carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes ChlZ and cytochrome b559 in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the ζ-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated π-electron systems and higher reduction potentials than β-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced β-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and β-zeacarotene (9 conjugated double bonds and 1 β-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the β-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl+/Car+ is higher in the mutant core complexes, consistent with the higher reduction potential for Car+. As the temperature increases, other carotenoids become accessible to oxidation by P680+.

Direct observation of the S1 level of the carotenoid spheroidene using near-infrared femtosecond spectroscopy

In this work, we have determined the energy of the S, state of the carotenoid spheroidene. The energy of this state is 13,400 ± 90 cm-1 at both 293 K and 186 K, showing that there is no temperature-induced shift of the SI level. A discrepancy of about 800 cm-1 between the S1 energy determined here and that obtained from previous fluorescence and resonance Raman measurements is explained in terms of the different conformational species co-existing in the S1 excited state.

The Effect of Solvent on the Lifetime of the Lowest Excited Singlet S1 (21Ag) State of Spheroidene

We are investigating the mechanisms by which carotenoids carry out their light-harvesting and photoprotective roles in photosynthesis. We are focusing on the carotenoid spheroidene (Fig. 1) which is found in both reaction center and antenna pigment-protein complexes from Rhodobacter sphaeroides wild type strain 2.4.1. A complete understanding of the spectroscopic and photochemical properties of spheroidene in vitro would be of substantial benefit in elucidating how it, and carotenoids in general, function in vivo. The effect of different solvent environments on the absorption, S0 → S2 (11Ag → 11Bu), spectra of carotenoids, including spheroidene, has been explored (1, 2). However, owing to the ultrafast lifetimes of the excited states of spheroidene, little is known about the effect different environments have on the dynamics of its excited states (3).