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Secondary acceptors

Bacterial reaction centers - the backreaction

Photosystem II - The two-electron gates for reduction of quinone

The two-electron gate of photosystem II and the mechanism of herbicide resistance

Structure of Photosystem II

The FeS centers of photosystem I

Donor reactions

Cytochrome c₂ as donor to bacterial reaction centers

Stereo view of the surface of cytochrome c₂ from Rb. sphaeroides, looking down on the heme cleft. Heme is magenta, hydrophobic residues are white, polar residues are green, acidic residues are red, basic residues are blue. Note the ring of basic residues forming the positively charged docking domain.

Stereo view of the surface of the reaction center from Rb. sphaeroides looking down from the periplasm. BChl is yellow, hydrophobic residues are white, polar residues are green, acidic residues are red, basic residues are blue. Note the ring of acidic residues forming the negatively charged docking domain.

As a consequence of this electrostatic binding, the reaction of cyt c₂ with the RC has the following characteristics:

• The cyt c₂ binds with an high affinity (K_D ~1µM at low ionic strength).
• At low ionic strength, and appropriate [cyt c₂], cyt oxidation following flash activation is first-order (reflecting the bound substrate). The reaction in vivo is also first-order, suggesting that at the local concentration and ionic strength, cyt c₂ is bound.
• At high ionic strength, and/or low [cyt c₂], the reaction is second-order, reflecting the reaction of the cyt from the unbound state.
• The second-order rate constant at low ionic strength is ~3 x 10⁸ M^-1 s^-1, but this value is strongly dependent on ionic strength.

The cytochrome c subunit of the Rps. viridis reaction center

Plastocyanin as donor to photosystem I

Stereo view of the surface of plastocyanin. Cu atom is hidden near top of molecule. Hydrophobic residues are white, polar residues are green, acidic residues are red, basic residues are blue. Note the two patches of acidic residues thought to form a negatively charged docking surface for interaction with cyt f or PS I.

The donor side of photosystem II - overview

Research on photosystem II and the mechanism of O₂-evolution spans a wide range of techniques and disciplines. Much recent work has concentrated on application of various spectroscopies to an exploration of the function and structure of the Mn-complex which is central to the water oxidation process. That work is covered briefly in Lecture 24. In this brief overview, we will consider biochemical and structural aspects.

Click here to review the structure through a Chime tour of a model of photosystem II.

The model structure contains only the D1 and D2 subunits of photosystem II, for which the L and M subunits of the bacterial reactions centers provides a template. The protein in the membrane is a more complex structure, with an antenna of chlorophyll binding proteins (CP43, CP47), additional chlorphyll proteins which interface with the bulk antenna of LHCII (CP24, CP26, CP29), two subunits of cytochrome b-559 (which may serve a role in photoprotection), three extrinsic proteins, the OEEP complex (Oxygen Evolution Enhancing Proteins,- 17, 22 and 33 Kd extrinsic subunits, shown as O, P, Q below), act as ionic concentrators to maintain a suitable ionic environment at the Mn-cluster, and a number of additional subunits of uncertain function.

(image taken from a nice review of the photosystems by Wei-Zhong He & Richard Malkin.

With the development of a relatively pure preparation of photosystem II (the BBY prep of Berthold, Babcock and Yoccum), which retained full activity for oxygen evolution, great progress has been made in understanding the biochemistry. The smallest complex which retains photochemical function consists of D1, D2, cyt b-559, and possible other small subunits. The smallest complex which retains a normal O₂-evolution contains in addition, CP43, CP47 (the core antenna), and the OEEP complex. The latter can be removed by various salt-washing treatments, and O₂-evolution at normal salt concentrations is lost, but can be largely restored by addition of Ca²⁺ and Cl^- at high concentrations.

Recently, progress has been reported on the crystallographic determination of structures for PS II. Kühlbrandt and colleagues, in collaboration with Barber's group, have reported a preliminary structure at about 8 Å resolution using electron diffraction of 2-D crystals. In combination with electron micrscopy of negatively stained images, and image averaging, they have generated a low resolution structure, which is summarized in the picture below:

From Barber J. (1998) Photosystem 2. Bioch. Biophys Acta 1365, 269-277.

A second effort is underway in Witt's group in Berlin, where preliminary reports of 3-D crystals, which diffract to better than 6 Å were discussed at the International Congress on Photosynthesis in Budapest (1998).

Y_Z, the secondary donor to P⁺₆₈₀

With the availability of sequences, and the application of molecular engineering to photosystem II, it was demonstrated that Signal II_s came from a redox active tyrosine-160 in D2 (Y_D), and Signal II_f (or II_vf) from tyrosine-161 in D1 (Y_Z). Time resolution in the ns range has now been achieved using near UV kinetic spectrophotometry, allowing resolution of the kinetics of oxidation and reduction of Y_Z, and a demonstration that the oxidation kinetics match the reduction of P⁺₆₈₀, and the reduction kinetics the oxidation of the S-states. The position of these residues in relation to the Mn-cluster and P₆₈₀ can be seen in the structural model of photosystem II. Further discussion of the spectroscopy of Y_Z can be found in Lecture 25.

Spectroscopy of Y_z

Difference spectra (Tyr^· - Tyr) in the near UV attributable to:

• Left: the free radical produced by UV irradiation of tyrosine, and
• the tyrosine free-radicals of photosystem II
  • Center: Tyr-160 of D2, Y_D,
  • Right: Tyr-161 of D1, Y_Z
  Note that the "derivative" band between 420 and 450 nm is due to a local electrochromic effect attributed to the charge on Y_Z⁺.

The kinetics of relaxation of the free-radical species after flash illumination.

• Left: Relaxation of Y_D⁺. Traces measured at the radical band (250 nm), and at the peak of the electrochromic band (430 nm), show the same kinetics.
• Right: Relaxation of Y_Z⁺. Traces measured at the radical band (245 nm), and at the peak of the electrochromic band (430 nm), show the same kinetics; the trace at 325 nm is due to the Q_A^-, and the kinetics show the electron transfer to Q_B. The backreaction measured here is that from Q_B^- to Y_Z⁺. (Note that the temperature is -8° C)

The EPR spectrum of Y_Z⁺.

Left: EPR spectra of a photosystem II core complex
• Top: Sample frozen in the dark before illumination
• Middle: Sample frozen under illumination to trap the Y_Z⁺ state
• Bottom: Illuminated sample, thawed and refrozen in the dark (to allow relaxation of the Y_Z⁺ state).

  Right: Spectra of deuterated samples

• Top: Same as middle, left
• Middle: Grown with b-methylene-3',3'-deuterated tyrosine
• Bottom: Grown with ring-3,5-deuterated tyrosine.

The Mn-complex

More about the spectroscopy of the Mn-complex can be found under Lecture 25.

Much information about the involvement of specific residues in binding Mn and/or Ca²⁺ at the active site has come from molecular engineering by mutagenesis. In D1, residue changes giving altered O₂-evolution or Mn-binding in loop ab (D59, D61, E65), loop cd (D170, E189, H190), and the C-terminal span (H332, E333, H337, D342, and the terminal -COO- of A344) identify potential ligands. Residue Y161 provides the secondary donor (Z), D170 is necessary for binding of Mn with high affinity in the assembly of the active OEC, but probably does not provide a ligand to the Mn-cluster, H198 is a possible ligand to the putative [Chl]₂, and H190 is essential for O₂-evolution, and a potential Mn-ligand. H195 as non-essential. These residues are in spans where the structure is unfortunately ill-defined by homology with the bacterial reaction centers. In D2, only E70 in loop ab looks like a good candidate.

See:
Debus, R. J. (1992) The manganese and calcium ions of photosynthetic oxygen evolution. Biochim Biophys Acta 1102, 269-352
for a comprehensive review

The intermediate electron transfer chains

The bc₁-complex

The b₆f-complex

The complex has the same complement of redox centers as the bc₁-complex, but the cytochrome b heme(s) for historical reasons are called cytochrome b₆, and the heme c center is called cytochrome f. The redox centers are bound in three subunits which show homology with the three redox subunits of the bc₁-complex. The protein corresponding to cytochrome b is separated into 2 subunits, with a "split" in the de loop.

• Cytochrome b, which shows strong homology with the N-terminal section of bacterial or mitochondrial cyt b, and shows a predicted folding pattern corresponding to the span from the N-terminus to the de span between transmembrane helices D and E. This segement contains both of the helices with putative histine ligands for the two hemes. Helix D has an extra residue inserted between the liganding histidines.
• The second part is called subunit IV (SUIV), and corresponds to the C-terminal part of bacterial cyt b, from the "split" in the de loop to a C-terminus truncated before the H-helix.

Structure

The structure of a soluble cytochrome f (which has lost the membrane anchor provided by the C-terminal helical span) has been solved at high resolution by Martinez, S.E., Smith, J.L., Huang, D., Szczepaniak, A. and Cramer, W.A.

Click here for a brief tour of the structure of cytochrome f, using Chime.

Mechanism

The mechanism of the b₆f-complex is still controversial. The experimental data can be interpreted as showing that the complex operates through a modified Q-cycle, just like the that for bc₁-complex, but with adjustments in rate constants and equilibrium constants as follows (Kramer and Crofts mechanism):

• The E_m values for cytochromes b_H and b_L are lower, with values of approx. -50 mV and -150 mV respectively.
• The semiquinone at the Q_i-site is much less stable (E_m < -50 mV for Q/SQ couple), so that reduction by cyt b_H does not occur significantly, and the site operates effectively by transferring two electrons only when both b-cytochromes are reduced.
• The complex turns over from the fully reduced state by an "autocatalytic" mechanism.

See:
Cramer, W.A., Soriano, G.M., Ponomarev, M., Huang, D., Zhang, H.,Martinez, S.E. and Smith, J.L. (1996) Some new structural aspects and old controversies concerning the cytochrome b₆ complex of oxygenic photosynthesis. Ann. Rev. Plant Physiol. Plant Mol. Biol. 47, 477-508.
for a recent review.

Ferredoxin and flavodoxin as mediators of electron flow from photosytem I to reductive metabolic pools

Electrons from photosystem I are delivered to NADPH, via a soluble redox protein,- either ferredoxin (in higher plants, and in algae under Fe-sufficient conditions), or flavodoxin (in algae under Fe-depleted conditions), and ferredoxin:NADP reductase (FNR).