Engineering Photosystem I for Enhanced Electron Transport Rates In ...
Engineering Photosystem I for Enhanced Electron Transport Rates In Vitro for Applied Photosynthesis Khoa Nguyen1,2, Natalie Myers3, Brendan Williams1, Danielle Harrell1 , Caitlin Paquet1& Barry D. Bruce1,2 1Department
of Biochemistry, Cellular, and Molecular Biology 2NSF STAIR (Sustainable Technology through Advanced Interdisciplinary Research) IGERT, University of Tennessee, Knoxville, TN 37996, USA 3School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA
Introduction
Solid-state, photovoltaic device ITO
AuPS IETL/C60 e Ag e -
e -
e -
Iwuchukwu I et al. Nature Nanotechnology 5, 73 - 79 (2010)
C. Lubner et al. Biochemistry 2010 49 (3), 404-414
-/+
B. Bruce
Recent projects in cyanobacteria has been the study Photosystem I and the utilization of its electron transport system. My current project is to engineer the PSI’s surface chemistry. For example, we are making complementary changes on both the PSI and cytochrome c6 surface so that they interact more quickly in solution. Specifically, we have made a psaF modifications that may allow more rapid docking of the electron donor, cytochrome c6 to the lumenal surface of PSI. After all modified PSI DNA constructs were made, they are required to be transformed into the organism and PSI can then be isolated from these transformants. These mutations will be functionally characterized through electrochemical and photochemical assays. The electron transfer rate can then be tested with a Pulsed LED Spectrometer (JTS-10) by measuring the re-reduction profile of P700. The kinetics of this electron transport step is being investigated as a function of cytochrome content, temperature, pH, ionic strength, and after selective mutagenesis. These results will be discussed in light of our understanding of how cyt c6 and PSI interact.
WT PSI
E
WT PSI
psaF Chlamy PSI
21.5kD
* BB
CC
(A) DNA construct used for electroporation. (B) and (C) represent a diagnostic PCR for psaF mutants. Amplification is achieved with a primer located 2300bp upstream of psaF and another located at the C terminus of psaF. In wild type T. elongatus, the resultant fragment is 2792bp, whereas the mutant fragment with the addition of 1200bp from the KanR gene as well as 249bp for the Chlamy insert sequence, results in a 4230bp fragment. However, it can be seen that in (B), the mutants contain both the desired mutant fragment, as well as the wild type fragment, which was verified via cloning subsequent sequencing. (C) represents the same diagnostic PCR following 11 weeks of segregation via single colony isolation and increasing the concentration of selection. (D) ,is a genomic map fragment representing the diagnostic PCR. (E), SDS gel showing the size of psaF in two wild type samples, but shifted up in the psaF-Chlamy mutant (F) Mass spec verification of psaF Chlamy modification via homologous recombination.
A D F 15237.627
6000
Wild Type PSI
psaD
5000
psaF
4000
psaL
3000
16133.194
2000 1000 0
15250.045
6000
psaF Chlamy Insert
4000 3000
psaF-absent
psaF Chlamy Insert
2000
16133.776
17979.039
1000 0
This project has stemmed off into many facets that involve various modifications/mutations to the subunits of PSI. DNA constructs of mutated psaF subunit gene have been made by a previous graduate student to increase PSI’s interaction with the electron donor cytochrome c6. The shown alignment and modification of T. elongatus psaF to Chlamydomonas reinhardtii psaF serve the purpose of increasing the cytochrome c6 interface at the F subunit, allowing for higher binding affinity. Along with increased binding affinity of cytochrome c6, platinum nanoclusters are also planned to be attached to the stromal side of the PSI complex, turning it into a hydrogen-evolving nanoparticle.
15000
15500
16000
16500
17000
17500
18000
*
*
14.4kD
5000
Design of T. elongatus with psaF-chlamy Gene
A
B
Confirmation of Transformed T. elongatus with psaF-chlamy Gene
18500
B
Intens. [a.u.]
A
10858.258
4000
3000
2000
Cyt 553 Mut\WT 11.18\3Lin
Wild Type Expected MW=10857
10931.614
3000 2500 2000 1500
(A) Samples of PSI complexes (3 μg chlorophyll/ ml) with cyt c6 (4 – 100 fold molar excess) were made in the presence of 2 mM ascorbate, 5mM MgCl2, 5mM MgSO4, 1mM MnCl2, 0.1mM Methyl viologen, and 20 mM MES buffer pH 6.4. Actinic flashes of 5ms (light source:Orange ring LED) were used at an intensity of 3,000 μE m-2 s-1. The Raw kinetic data was then fitted to an exponential two-phase association. (B) The observed rate constants were compared to cyt c6 concentration.
Effect of Temperature on P700+ Re-reduction by cyt c6 A
B
65°C 5°C
(A) Raw kinetic data of PSI /cyt c6 (10 fold molar excess) samples with measurement temperature ranging from 5oC-65oC. (B) The observed rate constants were compared to temperature (Kelvin).
Effect of Electrostatic Interactions on P700+ Re-reduction
Design and Confirmation of Mutated cyt c6 from T. elongatus Intens. [a.u.]
PSI-Pt nanocluster
PSI-Hydrogenase
Photosystem I has been shown to be a robust photoactive nanoparticle capable of generating both hydrogen and electricity in vitro. Both of these processes require the electrons to be transferred from between a soluble protein, such as plastocyanin and cytochrome c, to the oxidized special pair (P700) of the reaction center. Much of what we know about this process has been via the study of model photosynthetic prokaryotes such as cyanobacteria. These organisms are not only very important for global carbon fixation and oxygen production but are also powerful because they offer a robust and facile genetic system. Moreover, although they are prokaryotes they still possess oxygenic, two photosystem based process similar to what is found in higher plants. Recently, the thermophilic, rod-shaped, unicellular cyanobacterium, Thermosynechococcus elongatus BP-1 is of great interest in this field due to its fully sequenced genome and the success in crystallizing many highly thermostable protein complexes, including both PSI and PSII. An additional benefit of this organism is its ability to be genetically transformed via electroporation. Recently we have developed a genetic system to allow homologous recombination in cyanobacteria T. elongatus. Using this genetic approach we can manipulate individual PSI subunits by site-specific mutagenesis, adding epitope/affinity tags, and disrupting the gene products by insertional disruption. Using these molecular strategies we will create a portfolio of tools for improving the molecular properties of PSI such that it is more active as a hydrogenevolving nanoparticle.
Intens. [a.u.]
Applied Photosynthesis
Abstract
Intens. [a.u.]
Photosynthesis
Spectrophotometric Detection of P700+ Re-reduction by cyt c6
Cyt 553 Mut\Double 63,67 11.18\1Lin
Double 63,67 Expected MW=10929
A
B
1000 1000
10312.557
A
10914.667 4000
3000
Cyt 553 Mut\Single 63 11.18\1Lin
Single 63 Expected MW=10915
Intens. [a.u.]
Intens. [a.u.]
500 0
10868.603
Cyt 553 Mut\Single 67 11.18\1Lin
Absent heme
Single 67 Expected MW=10871 10257.239
Intens. [a.u.]
Intens. [a.u.]
Double 63,69 Expected MW=10916
Expected Laser flash has sufficient intensity with ns duration.
1000
0
1000
4000
Cyt 553 Mut\Double 63,69 11.18\1Lin
2000
1000
1500
10915.926
5000
3000
Absent heme
2000
Current LED flash with max intensity (3000µE)
0
0
10872.029
5000 4000 3000
Cyt 553 Mut\Double 67,69 11.18\1Lin
Double 67,69 Expected MW=10872
2000
B
0 x10 4
1.5
1.0
Single 69 Expected MW=10858
10861.823
Cyt 553 Mut\Single 69 11.18\1Lin
Intens. [a.u.]
1000
Intens. [a.u.]
(A) Mutations (yellow residues) were also made to T. elongatus cytochrome , in which acidic residues were introduced to complement the basic residues that were added to the psaF subunit. (B) Mass spec verification of cytochrome mutants
500
0
10926.179 6000
4000
0.5
Triple Expected MW=10929
2000
0.0
Cyt 553 Mut\Triple 11.18\1Lin
C
D Amplitude of Absorbance Change = Amount of oxidized P700
10308.321
0 8500
9000
9500
10000
10500
11000
11500
12000
12500
8500
9000
9500
10000
10500
m/z
11000
11500
12000
12500
(A) Wild type and psaF Chlamy PSI tested at two temperatures, in the presence of WT T. elongatus cyt. (B) psaF Chlamy PSI in the presence of Triple Mutant cyt. (C) psaF Chlamy P700+ re-reduction rates with WT cyt. (green), single mutants (red), and triple mutant (blue). (D) The flash durations tested at the two cytochrome concentrations shows the time required for complete P700 oxidation.
m/z
Spectrophotometric Detection of P700+ A
C PSI
Asc Cyt.C
P700+
Cyt.C6 FBMV
FBMV
P700
P700+
B
Using algal/plant models to modify psaF subunit of T. elongatus. A, Partial alignment of F subunit peptide sequences (N-terminus only) . B, Comparative view between T. elongatus and Spinach psaF subunit.
As expected, there was no significant change in the rereduction time between WT and psaF Chlamy PSI in the presence of WT cytochrome. However, in the presence of the triple mutant cytochrome, the re-reduction of psaF Chlamy PSI was much slower than expected. This suggests that the actinic flash duration is still on while the bound cyt has already donated its electron. The single mutants support this hypothesis with the least significant mutation (D67E) was the closest to WT and the most significant mutation (G63D) was the slowest of the three single mutants. In order to measure a single P700 to P700+ turnover, we must maintain flash intensity with a duration in the low µs range, achievable with a laser system (in progress).
1. Efficient homologous recombination in T.e. 2. Completed complementary mutagenesis of cyt c / PSI interface 3. Verification of mutations (PSI & cyt.c) by mass spec analysis 4. Measured P700 re-reduction kinetics 5. Kinetics depends on [cyt c] and temperature 6. Measured kinetics of P700 re-reduction using mutated PSI and mutant cytochromes 7. Test single turnover with laser system 8. Test improved yield of hydrogen evolution.
Acknowledgements
e-
N. Myers 2009
Conclusions & Future Work
e-
(A). Joliot Type Spectrometer (JTS-10): Diagram of the LED pump-probe spectrometer. Sample includes sodium ascorbate to reduce cytochrome C and methyl viologen as a final electron acceptor from FB (Fe-S cluster) (B). Sample full spectra of P700 recovery with detection sequence showing the average of three actinic flashes. (C). Depiction of monomeric PS I complex from T. elongatus with P700+ getting re-reduced by cytochrome after flash-induced photo-oxidation.
This work was supported by a National Science Foundation (NSF) Nanoscience Interdisciplinary Research Team (NIRT) award to B.D.B. (DBI-0403781); an NSF Sustainable Science Grant (CBET 0828615) award BDB; a SARIF Award to BDB. KN was supported by an IGERT:STAIR:Sustainable Technology through Advance Interdisciplinary Research (DGE-0801470) award to BDB and generous support from the Gibson Family Foundation and the Army Research Lab. Special thanks to Michael Vaughn and John Sonewald for assistance with the JTS-10.
of Biochemistry, Cellular, and Molecular Biology 2NSF STAIR (Sustainable Technology through Advanced Interdisciplinary Research) IGERT, University of Tennessee, Knoxville, TN 37996, USA 3School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA
Introduction
Solid-state, photovoltaic device ITO
AuPS IETL/C60 e Ag e -
e -
e -
Iwuchukwu I et al. Nature Nanotechnology 5, 73 - 79 (2010)
C. Lubner et al. Biochemistry 2010 49 (3), 404-414
-/+
B. Bruce
Recent projects in cyanobacteria has been the study Photosystem I and the utilization of its electron transport system. My current project is to engineer the PSI’s surface chemistry. For example, we are making complementary changes on both the PSI and cytochrome c6 surface so that they interact more quickly in solution. Specifically, we have made a psaF modifications that may allow more rapid docking of the electron donor, cytochrome c6 to the lumenal surface of PSI. After all modified PSI DNA constructs were made, they are required to be transformed into the organism and PSI can then be isolated from these transformants. These mutations will be functionally characterized through electrochemical and photochemical assays. The electron transfer rate can then be tested with a Pulsed LED Spectrometer (JTS-10) by measuring the re-reduction profile of P700. The kinetics of this electron transport step is being investigated as a function of cytochrome content, temperature, pH, ionic strength, and after selective mutagenesis. These results will be discussed in light of our understanding of how cyt c6 and PSI interact.
WT PSI
E
WT PSI
psaF Chlamy PSI
21.5kD
* BB
CC
(A) DNA construct used for electroporation. (B) and (C) represent a diagnostic PCR for psaF mutants. Amplification is achieved with a primer located 2300bp upstream of psaF and another located at the C terminus of psaF. In wild type T. elongatus, the resultant fragment is 2792bp, whereas the mutant fragment with the addition of 1200bp from the KanR gene as well as 249bp for the Chlamy insert sequence, results in a 4230bp fragment. However, it can be seen that in (B), the mutants contain both the desired mutant fragment, as well as the wild type fragment, which was verified via cloning subsequent sequencing. (C) represents the same diagnostic PCR following 11 weeks of segregation via single colony isolation and increasing the concentration of selection. (D) ,is a genomic map fragment representing the diagnostic PCR. (E), SDS gel showing the size of psaF in two wild type samples, but shifted up in the psaF-Chlamy mutant (F) Mass spec verification of psaF Chlamy modification via homologous recombination.
A D F 15237.627
6000
Wild Type PSI
psaD
5000
psaF
4000
psaL
3000
16133.194
2000 1000 0
15250.045
6000
psaF Chlamy Insert
4000 3000
psaF-absent
psaF Chlamy Insert
2000
16133.776
17979.039
1000 0
This project has stemmed off into many facets that involve various modifications/mutations to the subunits of PSI. DNA constructs of mutated psaF subunit gene have been made by a previous graduate student to increase PSI’s interaction with the electron donor cytochrome c6. The shown alignment and modification of T. elongatus psaF to Chlamydomonas reinhardtii psaF serve the purpose of increasing the cytochrome c6 interface at the F subunit, allowing for higher binding affinity. Along with increased binding affinity of cytochrome c6, platinum nanoclusters are also planned to be attached to the stromal side of the PSI complex, turning it into a hydrogen-evolving nanoparticle.
15000
15500
16000
16500
17000
17500
18000
*
*
14.4kD
5000
Design of T. elongatus with psaF-chlamy Gene
A
B
Confirmation of Transformed T. elongatus with psaF-chlamy Gene
18500
B
Intens. [a.u.]
A
10858.258
4000
3000
2000
Cyt 553 Mut\WT 11.18\3Lin
Wild Type Expected MW=10857
10931.614
3000 2500 2000 1500
(A) Samples of PSI complexes (3 μg chlorophyll/ ml) with cyt c6 (4 – 100 fold molar excess) were made in the presence of 2 mM ascorbate, 5mM MgCl2, 5mM MgSO4, 1mM MnCl2, 0.1mM Methyl viologen, and 20 mM MES buffer pH 6.4. Actinic flashes of 5ms (light source:Orange ring LED) were used at an intensity of 3,000 μE m-2 s-1. The Raw kinetic data was then fitted to an exponential two-phase association. (B) The observed rate constants were compared to cyt c6 concentration.
Effect of Temperature on P700+ Re-reduction by cyt c6 A
B
65°C 5°C
(A) Raw kinetic data of PSI /cyt c6 (10 fold molar excess) samples with measurement temperature ranging from 5oC-65oC. (B) The observed rate constants were compared to temperature (Kelvin).
Effect of Electrostatic Interactions on P700+ Re-reduction
Design and Confirmation of Mutated cyt c6 from T. elongatus Intens. [a.u.]
PSI-Pt nanocluster
PSI-Hydrogenase
Photosystem I has been shown to be a robust photoactive nanoparticle capable of generating both hydrogen and electricity in vitro. Both of these processes require the electrons to be transferred from between a soluble protein, such as plastocyanin and cytochrome c, to the oxidized special pair (P700) of the reaction center. Much of what we know about this process has been via the study of model photosynthetic prokaryotes such as cyanobacteria. These organisms are not only very important for global carbon fixation and oxygen production but are also powerful because they offer a robust and facile genetic system. Moreover, although they are prokaryotes they still possess oxygenic, two photosystem based process similar to what is found in higher plants. Recently, the thermophilic, rod-shaped, unicellular cyanobacterium, Thermosynechococcus elongatus BP-1 is of great interest in this field due to its fully sequenced genome and the success in crystallizing many highly thermostable protein complexes, including both PSI and PSII. An additional benefit of this organism is its ability to be genetically transformed via electroporation. Recently we have developed a genetic system to allow homologous recombination in cyanobacteria T. elongatus. Using this genetic approach we can manipulate individual PSI subunits by site-specific mutagenesis, adding epitope/affinity tags, and disrupting the gene products by insertional disruption. Using these molecular strategies we will create a portfolio of tools for improving the molecular properties of PSI such that it is more active as a hydrogenevolving nanoparticle.
Intens. [a.u.]
Applied Photosynthesis
Abstract
Intens. [a.u.]
Photosynthesis
Spectrophotometric Detection of P700+ Re-reduction by cyt c6
Cyt 553 Mut\Double 63,67 11.18\1Lin
Double 63,67 Expected MW=10929
A
B
1000 1000
10312.557
A
10914.667 4000
3000
Cyt 553 Mut\Single 63 11.18\1Lin
Single 63 Expected MW=10915
Intens. [a.u.]
Intens. [a.u.]
500 0
10868.603
Cyt 553 Mut\Single 67 11.18\1Lin
Absent heme
Single 67 Expected MW=10871 10257.239
Intens. [a.u.]
Intens. [a.u.]
Double 63,69 Expected MW=10916
Expected Laser flash has sufficient intensity with ns duration.
1000
0
1000
4000
Cyt 553 Mut\Double 63,69 11.18\1Lin
2000
1000
1500
10915.926
5000
3000
Absent heme
2000
Current LED flash with max intensity (3000µE)
0
0
10872.029
5000 4000 3000
Cyt 553 Mut\Double 67,69 11.18\1Lin
Double 67,69 Expected MW=10872
2000
B
0 x10 4
1.5
1.0
Single 69 Expected MW=10858
10861.823
Cyt 553 Mut\Single 69 11.18\1Lin
Intens. [a.u.]
1000
Intens. [a.u.]
(A) Mutations (yellow residues) were also made to T. elongatus cytochrome , in which acidic residues were introduced to complement the basic residues that were added to the psaF subunit. (B) Mass spec verification of cytochrome mutants
500
0
10926.179 6000
4000
0.5
Triple Expected MW=10929
2000
0.0
Cyt 553 Mut\Triple 11.18\1Lin
C
D Amplitude of Absorbance Change = Amount of oxidized P700
10308.321
0 8500
9000
9500
10000
10500
11000
11500
12000
12500
8500
9000
9500
10000
10500
m/z
11000
11500
12000
12500
(A) Wild type and psaF Chlamy PSI tested at two temperatures, in the presence of WT T. elongatus cyt. (B) psaF Chlamy PSI in the presence of Triple Mutant cyt. (C) psaF Chlamy P700+ re-reduction rates with WT cyt. (green), single mutants (red), and triple mutant (blue). (D) The flash durations tested at the two cytochrome concentrations shows the time required for complete P700 oxidation.
m/z
Spectrophotometric Detection of P700+ A
C PSI
Asc Cyt.C
P700+
Cyt.C6 FBMV
FBMV
P700
P700+
B
Using algal/plant models to modify psaF subunit of T. elongatus. A, Partial alignment of F subunit peptide sequences (N-terminus only) . B, Comparative view between T. elongatus and Spinach psaF subunit.
As expected, there was no significant change in the rereduction time between WT and psaF Chlamy PSI in the presence of WT cytochrome. However, in the presence of the triple mutant cytochrome, the re-reduction of psaF Chlamy PSI was much slower than expected. This suggests that the actinic flash duration is still on while the bound cyt has already donated its electron. The single mutants support this hypothesis with the least significant mutation (D67E) was the closest to WT and the most significant mutation (G63D) was the slowest of the three single mutants. In order to measure a single P700 to P700+ turnover, we must maintain flash intensity with a duration in the low µs range, achievable with a laser system (in progress).
1. Efficient homologous recombination in T.e. 2. Completed complementary mutagenesis of cyt c / PSI interface 3. Verification of mutations (PSI & cyt.c) by mass spec analysis 4. Measured P700 re-reduction kinetics 5. Kinetics depends on [cyt c] and temperature 6. Measured kinetics of P700 re-reduction using mutated PSI and mutant cytochromes 7. Test single turnover with laser system 8. Test improved yield of hydrogen evolution.
Acknowledgements
e-
N. Myers 2009
Conclusions & Future Work
e-
(A). Joliot Type Spectrometer (JTS-10): Diagram of the LED pump-probe spectrometer. Sample includes sodium ascorbate to reduce cytochrome C and methyl viologen as a final electron acceptor from FB (Fe-S cluster) (B). Sample full spectra of P700 recovery with detection sequence showing the average of three actinic flashes. (C). Depiction of monomeric PS I complex from T. elongatus with P700+ getting re-reduced by cytochrome after flash-induced photo-oxidation.
This work was supported by a National Science Foundation (NSF) Nanoscience Interdisciplinary Research Team (NIRT) award to B.D.B. (DBI-0403781); an NSF Sustainable Science Grant (CBET 0828615) award BDB; a SARIF Award to BDB. KN was supported by an IGERT:STAIR:Sustainable Technology through Advance Interdisciplinary Research (DGE-0801470) award to BDB and generous support from the Gibson Family Foundation and the Army Research Lab. Special thanks to Michael Vaughn and John Sonewald for assistance with the JTS-10.
