scholarly journals The Hydrophobic Recognition Site Formed by Residues PsaA-Trp651and PsaB-Trp627of Photosystem I inChlamydomonas reinhardtiiConfers Distinct Selectivity for Binding of Plastocyanin and Cytochromec6

2004 ◽  
Vol 279 (19) ◽  
pp. 20009-20017 ◽  
Author(s):  
Frederik Sommer ◽  
Friedel Drepper ◽  
Wolfgang Haehnel ◽  
Michael Hippler
Keyword(s):  
Electron Transfer ◽  
Dissociation Constant ◽  
Photosystem I ◽  
Tight Binding ◽  
Recognition Site ◽  
Site Directed Mutagenesis ◽  
Tryptophan Residue ◽  
Wild Type ◽  
Differential Selectivity ◽  
Electron Transfer Complex

On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp627is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochromec6to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002)J. Biol. Chem.277, 6573–6581). To examine the function of PsaA-Trp651in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation ofChlamydomonas reinhardtii.The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt)c6by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp651to Ser had an even larger impact on the dissociation constant. TheKDvalue increased another 2-fold when the values obtained for the interaction and electron transfer between cytc6and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in aKDvalue of about 554 μmthat is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp651is, such as PsaB-Trp627, crucial for high affinity binding of pc and cytc6to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp651and PsaB-Trp627confers a differential selectivity in binding of both donors to PSI.

scholarly journals The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex

2005 ◽  
Vol 280 (16) ◽  
pp. 16135-16142 ◽  
Author(s):  
Vadim Yu Kuznetsov ◽  
Emek Blair ◽  
Patrick J. Farmer ◽  
Thomas L. Poulos ◽  
Amanda Pifferitti ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Tight Binding ◽  
Electron Transfer Reaction ◽  
Wild Type ◽  
Transfer Rates ◽  
Marginal Change ◽  
Theoretical Predictions ◽  
Sulfur Protein ◽  
Key Residues ◽  
Electron Transfer Complex

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) fromPseudomonas putidawas studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 Å. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp38and Trp106, and at the edge of the protein-protein interface, Tyr33and Arg66, were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Δ106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Δ106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp106and Tyr33/Arg66substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr33, Arg66, and Trp106prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106but are likely to include Asp38and Cys39.

scholarly journals Essential Role of a Single Arginine of Photosystem I in Stabilizing the Electron Transfer Complex with Ferredoxin

2000 ◽  
Vol 275 (10) ◽  
pp. 7030-7036 ◽  
Author(s):  
Patrick Barth ◽  
Isabelle Guillouard ◽  
Pierre Sétif ◽  
Bernard Lagoutte
Keyword(s):  
Electron Transfer ◽  
Photosystem I ◽  
Essential Role ◽  
Electron Transfer Complex

scholarly journals Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

2001 ◽  
Vol 118 (5) ◽  
pp. 481-494 ◽  
Author(s):  
Li-Qun Gu ◽  
Stephen Cheley ◽  
Hagan Bayley
Keyword(s):  
Residence Time ◽  
Organic Molecules ◽  
Tight Binding ◽  
Major Effect ◽  
Site Directed Mutagenesis ◽  
Channel Blockers ◽  
Wild Type ◽  
Sensor Element ◽  
Kd Values ◽  
In The Wild

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼104-fold more avidly than the remaining αHL pores, including WT-αHL. The lower Kd values of these mutants are dominated by reduced values of koff. The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors.

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