Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from <i>Arabidopsis thaliana</i>

The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol–disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of r<i>At</i>PDI-A, the recombinant form of <i>Arabidopsis thaliana</i> PDI-A, have been investigated. As expressed, r<i>At</i>PDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron–sulfur (Fe–S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of r<i>At</i>PDI-A indicates that only the second and third cysteines of the CXX­X­C­KHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of r<i>At</i>PDI-A to accommodate an Fe–S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family

Structure-Based Exciton Hamiltonian and Dynamics for the Reconstituted Wild-type CP29 Protein Antenna Complex of the Photosystem II

We provide an analysis of the pigment composition of reconstituted wild type CP29 complexes. The obtained stoichiometry of 9 ± 0.6 Chls <i>a</i> and 3 ± 0.6 Chls <i>b</i> per complex, with some possible heterogeneity in the carotenoid binding, is in agreement with 9 Chls <i>a</i> and 3.5 Chls <i>b</i> revealed by the modeling of low-temperature optical spectra. We find that ∼50% of Chl <i>b</i>614 is lost during the reconstitution/purification procedure, whereas Chls <i>a</i> are almost fully retained. The excitonic structure and the nature of the low-energy (low-E) state(s) are addressed via simulations (using Redfield theory) of 5 K absorption and fluorescence/nonresonant hole-burned (NRHB) spectra obtained at different excitation/burning conditions. We show that, depending on laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different NRHB and emission spectra. The red-shifted state A near 682.4 nm is assigned to a minor (∼10%) subpopulation (sub. II) that most likely originates from an imperfect local folding occurring during protein reconstitution. Its lowest energy state A (localized on Chl <i>a</i>604) is easily burned with λ_B = 488.0 nm and has a red-shifted fluorescence origin band near 683.7 nm that is not observed in native (isolated) complexes. Prolonged burning by 488.0 nm light reveals a second low-E trap at 680.2 nm (state B) with a fluorescence origin band at ∼681 nm, which is also observed when using a direct low-fluence excitation near 650 nm. The latter state is mostly delocalized over the <i>a</i>611, <i>a</i>612, <i>a</i>615 Chl trimer and corresponds to the lowest energy state of the major (∼90%) subpopulation (sub. I) that exhibits a lower hole-burning quantum yield. Thus, we suggest that major sub. I correspond to the native folding of CP29, whereas the red shift of the Chl <i>a</i>604 site energy observed in the minor sub. II occurs only in reconstituted complexes

Trapping Dynamics in Photosystem I‑Light Harvesting Complex I of Higher Plants Is Governed by the Competition Between Excited State Diffusion from Low Energy States and Photochemical Charge Separation

The dynamics of excited state equilibration and primary photochemical trapping have been investigated in the photosystem I-light harvesting complex I isolated from spinach, by the complementary time-resolved fluorescence and transient absorption approaches. The combined analysis of the experimental data indicates that the excited state decay is described by lifetimes in the ranges of 12–16 ps, 32–36 ps, and 64–77 ps, for both detection methods, whereas faster components, having lifetimes of 550–780 fs and 4.2–5.2 ps, are resolved only by transient absorption. A unified model capable of describing both the fluorescence and the absorption dynamics has been developed. From this model it appears that the majority of excited state equilibration between the bulk of the antenna pigments and the reaction center occurs in less than 2 ps, that the primary charge separated state is populated in ∼4 ps, and that the charge stabilization by electron transfer is completed in ∼70 ps. Energy equilibration dynamics associated with the long wavelength absorbing/emitting forms harbored by the PSI external antenna are also characterized by a time mean lifetime of ∼75 ps, thus overlapping with radical pair charge stabilization reactions. Even in the presence of a kinetic bottleneck for energy equilibration, the excited state dynamics are shown to be principally trap-limited. However, direct excitation of the low energy chlorophyll forms is predicted to lengthen significantly (∼2-folds) the average trapping time