Why study photosynthesis? Largest biochemical process on earth. Source of earth’s atmospheric oxygen (began 1.5-2.0 billion years ago). Source of nearly all biologically useful energy. Crystal structures known for each membrane protein complex (several Nobel Prizes). Light is the substrate and ultra-fast kinetic events can be studied under extremely short time periods. Intellectually challenging and complex (biophysics, biochemistry, molecular biology, ecology).

6C(H2O) + 6O2 + 18 ADP + 18Pi + 12 NADP + 6H2O The Dark Reactions 6CO2 + 12H2O + 18ATP + 12NADPH  6C(H2O) + 6O2 + 18 ADP + 18Pi + 12 NADP + 6H2O The Light Reactions

Two Types of Reaction Centers

Michel, Deisenhoffer and Huber - Nobel Prize in Chemistry, 1989 Rhodobacter viridis Photosynthetic Bacterial Reaction Center Non-oxygenic Photosynthesis First membrane protein structure resolved at atomic levels of resolution by X-ray diffraction. Michel, Deisenhoffer and Huber - Nobel Prize in Chemistry, 1989  

The R. viridis BRC is a type-II reaction center L subunit- Binds chlorophyll, pheophytin and quinone cofactors involved in electron transfer M subunit – Binds chlorophyll, pheophytin and quinone cofactors involved in electron transfer H subunit – Stabilizes complex Cyt C – extrinsic protein binds four hemes involved in electron transfer Cofactors: 4 hemes, Chlsp, 2 Chlm, 2 Pheophytin, 2 quinones, 1 non-heme Fe.

Pigments in photosynthesis Beta carotene

Quinones in photosynthesis mobile and fixed, electron and proton donors and acceptors

Cofactor orientation and energetics in the BRC Energy Level Chlsp +0.45 eV eV = 0.7 Qa -0.2 eV

Protein-cofactor interactions Cofactor Ligands Chlsp L-H173, M-H200 Chlm L-H153, M-H180 Fe L-H190, L-H230, M-H217, M-H264 Carotenoid No residues involved in coordination Cyt C hemes 17 amino acid helix followed by a turn and Cys-X-Y-Cys-His. Hemes bound by thioether bond to Cys.

Function of the BRC in Photosynthesis A light driven proton pump working in concert with the cytochrome bc1 complex to generate a proton gradient and ATP.   Menaquinone in the QA site is singly reduced by an electron initially derived from the ChlSP (primary electron donor). Ubiquinone in the QB site is then sequentially reduced (2 e-) and protonated (2H+ ) via QA forming UQH2. UQH2 then exits from the QB binding pocket as a mobile 2 electron and 2 proton carrier and is oxidized by the cytochrome bc1 complex. The result is the transfer of protons across membrane to establish a pH gradient that drives ATP synthesis. 

Bacterial Photosynthesis

Oxygenic Photosynthesis

Linear photosynthetic electron transfer chain of oxygenic photosynthesis

Lateral heterogeneity of membrane protein complexes

Mobile electron carriers Plastocyanin Cu +2

Mobile electron carriers - ferredoxin

Photosystem II Model

D1 - D2 Proteins: cofactors and amino acid ligands Fe – D1-H215, D1-H272 D2-H214, D2-H268 ChlZ – D1-H118 ChlZ – D2-H117 YZ – D1-Y161 YD – D2-Y160 ChlSP – D1-H198, D2-H197

Organization of cofactors in the PSII RC psuedo-C2 symmetry Ferreira et al. (2004) Science 303: 1831

Relationship between the PSII RC and the proximal antennae complexes

 eV ChlZ cycle Charge Stabilization

P680+ is a very strong oxidant

Period 4 oscillation of oxygen evolution following single-turnover flashes 2H2O + 8hv  4H+ + 4e- + O2 Pierre Joliot

Kok’s clock, S-state transitions H+ H+

Metallo-radical model for water oxidation

PSI Structure

The PSI RC polypeptides also function as proximal antennae complexes psaA and psaB RC proteins are large - 81 kD. N-terminal six transmembrane spans bind the proximal antennae Chls analogous to the PSII CP43 and CP47 proteins (43-47 kD). Five, C-terminal transmembrane spans bind the reaction center cofactors analogous to the PSII D1 and D2 proteins (32 kD). Since PSII is very sensitive to photodamage and proteolytic turnover, unlike PSI, it is more efficient to repair only the damaged protein (D1) than the D1 and CP-43 protein. As a corollary, since photodamage is rare in PSI. its unnecessary for split proteins to facilitate repair

Photosystem I RC cofactors

PSI redox potential (energy level) and kinetics

Cytochrome b6f complex Cyt f = 32 kD, heme Cyt b6 = 24 kD, 2 hemes with different Em Rieske Fe-S = 19 kD, nuclear gene, Fe-His ligands shift Em of 2Fe-2S (+) Subunit IV = 17 kD, analogous to C-terminus of mitochondrial Cyt b PetG, PetL and PetM = 3-4 kD, ?

Chlamydomonas Cytochrome b6f structure Stroebel et al., (2003) Nature 426: 413

Chlamydomonas cytochrome b6f complex contains an unexpected c-type heme near the high-potential heme The novel heme may control access to the Qi site or participate in cyclic electron transfer between photosystem I

FeS head group moves between Qo site and heme c1,of analogous Cytochrome bc Complex Qi bH Qo bL FeS c1

Q-Cycle oxidant-induced reduction PQH2 = 0.0 eV PQ- = - 0.2 eV (PQ- more negative than PQH2 !!!) Qn heme = - 0.05 eV Qp heme = - 0.15 eV Rieske 2Fe-2S = + 0.3 eV Cyt f heme = + 0.34 eV PC Cu+2 = + 0.365 eV Rate PQH2 ox at Qp = 10 - 20 ms (rate limiting step in Ps) Rate PQ red at Qn = 0.1 ms