Introduction
Statement of Intent
Since the group has been successful with a rhenium 4,7-dimethyl-1,10-phenathroline tricarbonyl histidine modified azurine with a single reactive tryptophan. Therefore, if a viable hopping system is constructed, the desired back electron transfer should be greater than the previously reported 100-fold increase in the electron transfer rate.
Semiclassical Electron Transfer Theory
The kinetic parameters are reaction temperature, reaction driving force (–ΔG°), reorganization energy (λ), and electronic coupling matrix element (HAB). In each of the cases shown in Figure 1.1, the system is adiabatic since the product and reactants are located in the same nuclear coordinates.
Metal Modified Metalloproteins and Electron Transfer
With the -ΔG° = λ systems,23,25, the electron transfer rates depend on the HAB and therefore also the distance decay factor, β (see Figure 1.3). Also significant to the tunnel model is the broad distribution of electron transfer rates within.
Multi-Step Tunneling
For the transient absorption (figure 3.5), the copper oxidation signal has decreased so that no definite copper oxidation can be determined from the data. To compare the unusual biexponential emission decay observed for the oxidized sample (see figure 5.1), the same experiments were performed with the wild-type protein.
Materials and Methods
Photochemistry with Metalloproteins
The signal was not great for Cu2+ generation, however with forced fitting (see Figure 5.4) the rate was found to be about 5 x 106 s-1. Second, although the two proteins are structurally superimposed (see Figure 6.2), the ruthenium label can be oriented in a different configuration that slightly extends the distance between the electron transfer partners.
Appendix of Terms
Protein Preparation
However, samples of the oxidized protein (prepared with Cu2+ instead of Cu+) did show a concentration dependence of the emission lifetime (not shown). Using the tunneling pathway model, the β-coupling decay constant for β-sheets was predicted to be 1.1 Å-1, lower than the 1.4 Å-1 suggested by Dutton for proteins.5-8 Experimentally, azurin, a β-barrel protein, was analyzed for distance dependence of electron transfer1,6,7,9,10 and found to have a β-coupling decay constant of approximately 1.1 Å-1, as predicted by the tunneling pathway model.
Wavelength-Dependent Nanosecond Laser System (NS-1)
Time-resolved Infrared Spectroscopy (TRIR)
Each of the dmp ligands has a weak aromatic interaction with the neighboring tryptophan, similar to the stronger spread π stacking observed in the Re(H124)W122 crystal. The other striking feature of the crystal structure is the spacing between the rhenium and copper atoms. The pumping/purging started with a rapid draw of air in the headspace of the cuvette.
Inter-molecular Hopping through Dimers
Abstract
As seen in the luminescence decays (Figure 3.4) in the first series of laser experiments, the external reducing agent, sodium dithionite, disrupts the system. Re(H126)W122 solidified the importance of the communication between Re-label and the tryptophan intermediate for direct photooxidation. The luminescence decay for Re(H107)W108W110 is bi-exponential for both oxidized and reduced proteins and mono-exponential for the reduced protein in the presence of the oxidative quencher.
Designing a Hopping System
Experimental Results
The luminescence decay lifetime of the system with dithionite is about 300 ns and shows no concentration dependence. Due to the instability of the protein at high concentrations and low pH, a condition utilizing both high pH buffers for the well and protein solutions was chosen. Since some of these bands overlap and shift as a function of time, the time dependences of the band areas (obtained from Gaussian analysis of the spectra) were used for the kinetic fit.
Discussion
Unlike most relabeled azurin crystals, rhenium and its ligands do not interact with the rhenium label of the neighboring protein. With back electron transfer decay rates from both TRIR and early transient absorption data, distances extrapolated from the crystal structure indicate that intermolecular back electron transfer across the dimer interface is the most likely candidate to calculate the kinetics. data. With 50 ns luminescence quenching and 4 – 6 µs decay of the copper signal, the NS-1 data without sodium dithionite mirror the results seen for the CS state and the TRIR data.
Conclusions
Finally, the lifetime of the exponential decay of the reduced protein in the presence of 10 mM ruthenium(III) hexaamine oxidative quencher is 80 ns. According to the luminescence and transient absorption results of reduced Re(H83)W48W110, there was no formation of Cu2+. In the case of two similar proteins examined in this study, the structural modification is a covalent thioether linkage from the porphyrin to the peptide backbone.
Intra-molecular Hopping with Two Intermediates
Abstract
The mutant Re(H126)W124W122 uses three electron transfer steps to oxidize copper from the rhenium-excited state. Experiments are also provided that examine the environment of the tryptophan and the structure of the metal-modified protein.
Improving Intra-molecular Hopping with Tryptophan Wires
To separate the dimer interface and to provide a tight communication between the rhenium label and tryptophan, an additional tryptophan was inserted into the beta sheet. By placing a tryptophan (W124) next to Re, we were more likely to see efficient quenching of the Re excited state. Although the placement of a tryptophan at position 124 alone ensured an increase in efficient *Re quenching, we had only seen efficient copper oxidation in any of the engineered mare models when a tryptophan had been placed at position 122.
Experimental Results
Steady state fluorescence measurements were performed as described in Chapter 3.3, except with the addition of a temperature-controlled system. For the steady state fluorescence experiments, the samples were prepared by the same method used for the temperature dependent steady state experiments as well as in a buffer without the cryoprotectant. Steady state fluorescence was recorded at 25 °C for both glycerol and regular buffer and at -19 °C in glycerol buffer.
Discussion
With zinc Re(H126)W124W122, any quenching of rhenium luminescence would only be due to efficient quenching by the neighboring tryptophan 124. In the case of the double mutant, not only is dimethyl phenanthroline T-bonded to tryptophan 124, but both tryptophans share the same T-shaped π interaction. This effect is likely due to a strong interaction of dimethyl phenanthroline with the internal tryptophan 124 versus a neighboring tryptophan at an interface.
Conclusions
The distinct lack of fast decay in the reduced species (i.e. monoexponential decay) would mean that radical formation is an unlikely scenario. Smaller couplings were found for α-helices, corresponding to the prediction of a coupling decay constant β of about 1.26 Å-1. More importantly, site 92 can be used to determine the effect of the covalent thioether bond on the electron transfer pathway.
Double Tryptophan Mutants in a Hydrophobic Environment
Abstract
The desired goal was to use multiple electron steps to increase the rate of electron transfer before the loss of the proton present in the initial oxidized tryptophan intermediate. Because this proton loss can increase due to hydrophobic environments, only well-coupled, fast electron transfer can prevent a proton loss.
Attempting a Hydrophobic Hopping System with Tryptophan
In these cases, the kinetics favored a fast electron transfer more than the slower deprotonation step. Biological systems often favor kinetically driven hopping or a proton-coupled electron transfer (PCET) 2–4 to avoid a deprotonation step acting as a thermodynamic sink. Increased coupling between each redox site (metal or Trp) will enable a multi-step electron transfer that will be faster than the proton loss at any site.
Experimental Results
While neither mutant nor the wild type generated any Cu2+ signal with only reduced protein, the wild type protein did generate a small amount of Cu2+ with oxidative quencher (see figure 5.4). With the fixed parameters, IGOR was able to generate the fit (seen in figure 5.4 as a red line), which corresponds to a rate constant of 5 x 106 s-1. Since no significant transient absorption signal was recorded for the reduced species on its own, the oxidative quencher was added and the transient absorption analyzed (see figure 5.6).
Discussion
Anyway, the two decay lifetimes present in the oxidized samples are similar for both the mutant and the wild-type, so no new features (such as hopping) selectively occur in the mutant. The discrepancy in the short decay amplitudes between the oxidized and reduced species would indicate that either the short decay rate does not correspond to a faster extinction species (such as the initial W108 radical or the second W110 radical) or that the relative populations with rhenium orientation changed based on of the copper charge. As for the transient absorption observed in the presence of oxidative quencher (see Figure 5.7), no Cu+2 formation is present.
Conclusions
If the same principle is applied to the tryptophan double mutant, W110 is not generated due to the fact that the neutral radical W108 has a lower reduction potential than W110. Back electron transfer rates were determined for two ruthenium-modified cytochrome cb562 proteins. Since heme is the electron acceptor for electron transfer, the small structural change should be more important than most small structural changes in the protein.
Modifying Tunneling Pathways by Altering Protein Structural Features
In recent studies16,17 on experimental rate constants of azurin, cytochrome c, myoglobin and cytochrome b562, Beratan evaluated how the effects of the electron pathway affected rate. This issue will be addressed by determining ET rates in cytochrome cb562 mutants where the heme is covalently linked to the α-helical backbone. With the covalent attachment of the heme to the protein backbone, the tunneling pathway for each mutant can be restricted to a single, well-coupled pathway, or access a multiple pathway by providing greater access to the heme, the electron acceptor.
Materials and Methods
After sonication, the lysed cells were spun down to separate cell particles from the protein-containing periplasmic supernatant. Protein sample buffer was exchanged using a HiPrep 26/10 desalting column into 10 mM NaOAc pH 4.5. After purification on a Mono Q column, the protein was again exchanged with 10 mM NaOAc pH 4.1 buffer.
Experimental Results
The cuvette was attached to the Schlenk line and the Teflon seal was turned to the first seal (isolate the system from external air). As the Soret band blue shifts for the ferrous to ferric back electron transfer, there will be a bleach recovery at 409 nm (see figure 6.4) and a growth decay at 424 nm. This bleach recovery is indicated in Figure 6.4 as a positive first differential that continues in the range around 370 nm.
Discussion
D73H, had electron tunneling pathways that coupled to the metal acceptor, the iron heme, through one of the axial ligands. The remaining seven sites (one being the native histidine at position 63) were considered to have multiple pathways and coupled to the acceptor through the porphyrin ring. This increase in distance for a single dominant identically bonded system should lead to a slight decrease in the electron transfer rate.
![Table 6.2: Comparing Experimental Data to Theoretical Data [s -1 ]](https://thumb-ap.123doks.com/thumbv2/123dok/10401606.0/114.918.201.799.755.1018/table-6-2-comparing-experimental-data-theoretical-data.webp)
Conclusions
First, site 92 is on the fourth helix and in predictive models for the b562 protein, the surface of the porphyrin through which the electron travels is the same as the surface covalently linked in cb562. Since the electron transfer rates for both cysteine (K19C and K92C) cb562 mutants have already been determined by a fellow Gray group student, Nicole Bouley Ford, the electron transfer rates of histidine mutants with rigid metal labels can be compared to cysteine mutants with flexible metal labels. Investigation of electron transfer in the alpha-helical protein cytochrome b562, Thesis, California Institute of Technology, 1999.
Marcus Equation: Rate of Electron Transfer
Marcus Equation: Electronic Coupling
Tunneling Pathway: H AB proportionality
