Plastocyanin mutant

The electron-transfer kinetics of PSI-200 particles cross-linked with wild-type and mutant plastocyanins were examined by flash-induced absorbance changes. PSI-200 particles cross-linked with wild-type PC, shown in Fig. 8, trace (a), displays two P700 re-reduction phases with ty, of 13 jus and 141 /js, respectively, comparable to those found in vivo. For PSI-200 cross-linked with a mutant PC with modified residues 42 to 45 in the southern acidic patch, i.e., Asp-42->Asn, Glu-43->Gln, Asp-44->Asn and Glu-45->Gln, the rate of P700 re-reduction is dramatically retarded, as shown in Fig. 8, trace (b), giving two kinetic phases with ty of 160 //y and 766 /zy. When a mutant PC in which only one residue in the northern acidic patch was mutated, i.e., Glu-59->Gln, the resulting complex with PSI-200 yielded flash-induced P700 re-reduction kinetics which were almost the same as that for the wild-type plastocyanin, with ty, values of 13 /zy and 149 /js, as shown in Fig. 8, trace (c). When all three acidic residues in the northern acidic patch were converted to neutral ones, i.e., Glu-59->Gln, Glu-60->Gln and Asp-6l->Asn, the rapid re-reduction phase still retained a ty, of 14 /zy [Fig. 8, trace (d)]. 

Fig. 8. Re-reduction Kinetics of P700 in the compiexes formed between PSi-200 particies with wild-type and mutant plastocyanins using /V-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC) as the cross-linking agent. Nd-YAG laser flashes were used for excitation. The kinetic traces were presented on divided time scales of 0.2 and 2 ms per division, respectively, (a) PSI/wild-type PC (b), (c) and (d) PSI/mutant PC complexes (see legend on the right side for details). Decay time constants r.,sare also listed in the legend at right. Figure and data source Hippier, Reichert, Sutter, Zak, Altschmied, SchrOer, Herrmann and Haehnel (1996) The plastocyanin binding domain of photosystem I. EMBO J 15 6378.

PS1 The PS 1-prep, introduced in this communication is the first reported with a polyhistidine tag fused to the N-terminus of the PsaF subunit. This construct was possible due to the fact that cyanobacterial PsaF-deletion mutants show no impact on photoautotrophic growth - in contrast to Chlamydomonas reinhardtii, where inactivation of PsaF results in a severe reduction of electron transfer from plastocyanin to PS 1 [Hippier et al. 1997], Also, the N-terminus of the F-subunit which was decorated by the tag is located towards the lumen side which enables an attachment of the isolated PS1 with the lumen-exposed /donor-side to the electrode surface in our hydrogen-producing device. 

The electron donor to Chl+ in PSI of chloroplasts is the copper protein plastocyanin (Fig. 2-16). However, in some algae either plastocyanin or a cytochrome c can serve, depending upon the availability of copper or iron.345 Both QA and QB of PSI are phylloquinone in cyanobacteria but are plastoquinone-9 in chloroplasts. Mutant cyanobacteria, in which the pathway of phylloquinone synthesis is blocked, incorporate plasto-quinone-9 into the A-site.345a Plastoquinone has the structure shown in Fig. 15-24 with nine isoprenoid units in the side chain. Spinach chloroplasts also contain at least six other plastoquinones. Plastoquino-nes C, which are hydroxylated in side-chain positions, are widely distributed. In plastoquinones B these hydroxyl groups are acylated. Many other modifications exist including variations in the number of iso-prene units in the side chains.358 359 There are about five molecules of plastoquinone for each reaction center, and plastoquinones may serve as a kind of electron buffer between the two photosynthetic systems. 

Intriguingly, the blue copper sites, especiaUy those with a carbonyl oxygen at the axial coordination position, display high affinity for Zn + ions. Mutants in which the Met is replaced by Gin or Glu preferentiaUy bind Zn + when expressed in heterologous systems, e.g., Escherichia coli. Examples include azurin, amicyanin, nitrite reductase, and possibly also plastocyanin (Diederix et al., 2000 Hibino et al., 1995 Murphy et al., 1995 Nar et al., 1992a Romero et al., 1993). In the case of azurin it has been shown that both wild-type and the Met—Gin mutant have the same affinity for both Zn +and Cu + (Romero ci a/., 1993). In addition, EXAFS studies showed that some preparations of blue copper proteins purihed from their natural sources also contain small fractions of Zn derivatives (DeBeer George, personal communication). 

IB3I Plastocyanin T2S mutant) Prochlorothrix hollandka Babu et al., 1999... 

Rates of Cu+ to Ru + electron transfer also have been measured in modified mutants of spinach plastocyanin, a blue copper protein from the photosynthetic ET chain [79], Ru-bipyridine complexes were introduced at surface sites, with Cu-Ru distances ranging from 13 to 24 A. ET rate constants, measured using laser flash-quench techniques, vary from 10" to 10 s. ET in Ru-modified plastocyanin is not activationless as it is in Ru-modified azurin, suggesting a slightly greater reorganization energy for the photosynthetic protein. The distance dependence of ET in Ru-modified plastocyanin is exponential with a distance decay factor identical with that reported for Ru-modified azurin (1.1 A ). 

On the basis of the electronic, resonance Raman, and EPR spectra, the cysteine-containing copper proteins have been divided into four groups axial type 1 (e.g. plastocyanin), rhombic type 1 (e.g. nitrite reductase and stellacyanin), type 1.5, and type 2 (mutant) copper proteins [81]. We have studied the spectra of members of each group with the CASPT2 method [33,34,36,38]. 

Scanning probe microscopy characterization of gold-chemisorbed poplar plastocyanin mutants. Surface Science, 530, 181-194. 

Chitnis, Reilly and Nelson obtained a mutant of Synechocystis sp. PCC 6803 that is devoid of PsaE and, although incapable of autotrophic growth, can grow normally in the presence of glucose. The PS-I RC complex prepared from this mutant could not photoreduce Ed but could mediate electron transfer from plastocyanin to artificial electron acceptors such as methyl viologen. Thus PsaE is demonstrated to promote and enhance the electron transfer between [EeS-A/B] and Ed. 

S Modi, M Nordling, LG Lundberg, Hansson and DS Bendall (1992) Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin. Biochim Biophys Acta 1102 85-90... 

Battistuzzi G, Borsari M, Loschi L, Menziani MC, De Rienzo F, Sola M (2001) Control of metalloprotein reduction potential the role of electrostatic and solvation effects probed on plastocyanin mutants. Biochemistry 40 6422-6430... 

As mentioned earlier in this section, three of the four active site ligands in cupredoxins are situated close together on a single loop (see Fig. 5). In Table III the loops found in this area of three different cupredoxins are shown. To investigate the role the length and composition of this loop have on the active site properties of amicyanin the corresponding loops from plastocyanin (113) and azurin (130) have been introduced. The properties of these mutants are compared to wild-type protein in Table IV. In both cases a novel site that is produced does not resemble, spectroscopically, wild-type amicyanin or... 

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