proteins
Up to this point the model for the formation of an encounter complex and several methods for the analysis of such complexes have been de-scribed. To give an overview of the proteins subject of this thesis, the individual proteins will be highlighted in the following section. The detailed structure of Pc and Cytc6, two mobile proteins which play an important role in the electron transfer during photosynthesis, as well as the structure of Cytf, one of their reaction partners, is described.
Following to that, the complexes formed by these proteins are high-lighted.
Plastocyanin
Pc is the main interaction partner of Cytf and serves as an electron carrier to the chlorophyll of PSI.52 The length of Pc varies from 97 to 105 amino acids, depending on the organism, and forms a secondary structure consisting of seven β-sheets. The variants with up to 105 amino acids mainly occur in cyanobacteria. Pc belongs to the type I copper protein family, containing a redox active copper, coordinated by two histidines, one methionine and one cystein. The protein has
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Figure 4.1. The structure of Pc consists of seven β-sheets and has a copper atom as a cofactor. Pc has two significant regions, the hydrophobic and the basic/acidic (eastern) patch, which play an important role for complex for-mation with Cytf. The electrostatic potential for the organismNostoc, Phor-midiumand Poplar are mapped on the surface ranging from a -3 kBT/e0(red) to 3 kBT/e0(blue).
a characteristic blueish color at 597 nm, which is responsible for its alias ’blue copper protein’. The copper atom is buried 5 Å below the surface at a region mainly consisting of hydrophobic amino acids and therefore known as hydrophobic patch.53,54In Figure 4.1 an overview for the structural regions of Pc is given. PC includes a slightly charged region at the side of a hydrophobic patch which is commonly known as eastern patch. This region plays a major role in the complex formation procedure with Cytf.40,55In plants and the cyanobacteriumNostocsp.
PCC 7119 the eastern patch is complementary charged to Cytf. This regions contribute highly to the electrostatically stabilization of the complex formation and to the relative protein orientation within the complex.56,57For the thermophilicPhormidium laminosum, where the
eastern patch is less charged, the binding orientation is different. The low presence of electrostatic interaction causes the transient complex in Phormidium to be much more dynamic than in plants orNostoc.
Cytochrome c
6Cytc6 is the second electron transfer protein interacting with the cy-tochrome b6f complex. It is suggested, that depending on the envi-ronmental factors the bacteria switches for the electron transfer re-action from Pc to Cytc6 especially when there is an deficiency of cop-per.58,59Cytc6has a similar redox potential as Pc ranging from 335-390 mV,60,61and is therefore well suited to substitute Pc as an electron car-rier from Cytf complex to PSI.60,62 In higher plants a Cytc6 analogue was found, that has a lower redox potential.63 This makes it unsuited for the oxidation of Cytf and is hence presumably not involved in pho-tosynthesis.64 The structure of Cytc6is well studied and several crystal structures from various organisms including cyanobacteria, algae and terrestrial plants are known.65–67 The length of Cytc6 ranges from 83 to 90 amino acids and its secondary structure consists mainly of α
-Figure 4.2. Cytochromec6 is a globular shaped protein consisting mainly of α-helices. The cofactor heme is shown in red, the protein backbone in gray.
helices forming a ternary structure of globular shape. An overview of the Cytc6 structure is shown in Figure 4.2. Cytc6 belongs to the class I cytochromecfamily, where a single cytochromeccofactor is bound to the protein by a CXXCH motive with the fifth and sixth residues coor-dinating the heme-iron being histidine and methionine. Considering the primary and secondary structure few parallels can be seen to Pc.
In spite of the structural differences, Cytc6 and Pc show a similar iso-electric point.68 The interaction of Cytc6 with Nostoc Cytf is slightly reduced, compared to the one of Pc, which can be explained by the charges being regionally not as highly concentrated as for Pc.
Cytochrome f
Cytf is part of the cytochromeb6f complex, a homo-dimeric transmem-brane protein, located in the thylakoid memtransmem-brane in chloroplasts of plants, algae and cyanobacteria.69,70Cytf is the soluble part of the cyto-chromeb6f complex anchored to the transmembrane part of the com-plex by a helix of 30 hydrophobic amino acids. An overview of the cytochrome b6f complex with Cytf highlighted in red can be seen in Figure 4.3. As mentioned before, Cytf is the direct interaction partner of Pc or Cytc6, which transfers the electron yielded by Cytf to the PSI complex.52 The protein has a molecular weight of about 28 kDa and an elongated shape, which is directed along the thylakoid membrane.
Macroscopically Cytf consists of two domains, a big and a small one.71 Just like Cytc6, Cytf belongs to the c-type cytochrome family with the c-type heme bound in the big domain.72 This heme acts as the elec-tron donor for its redox partner and thus the region surrounding the heme is the active site for an electron transfer. The cofactor is cova-lently bound to the protein by two thioethers and the heme iron is additionally coordinated by a histidine and the N-terminus of the pro-tein chain.73 The heme is buried in a hydrophobic region at the edge between the two domains of Cytf, which represents one of the bind-ing sites for Pc and Cytc6.72 At the upper ridge of the small domain, an acidic or basic patch is located, which is electrostatically stabilizing the
Figure 4.3. Overview of the homo-dimeric cytochrome b6f complex an-chored to the thylakoid membrane. The complex is colored according its pro-tein chains. The cofactors bound to cytochromeb6f are highlighted in yellow.
The soluble part Cytf (red) is located on the thylakoid side. In Cytf the region around the c-type heme (black) binding site is called hydrophobic patch. The small protein domain has a region, that is differently charged depending on the organism also known as acidic or basic patch.
complex formation of Pc and Cytf. As well as in Pc the charged regions of the proteins vary depending on the organism. In detail, while the cyanobacterium Nostoc has a negatively charged patch in the small domain of Cytf and a positive one in the opposing counterpart, the same region in plants is charged contrariwise. A special role has Cytf in Phormidium, also a cyanobacterium, where in both proteins, Cytf and Pc, highly charged regions are lacking.