Abstract
The flash-quench method is developed to produce long-lived potent reactants.
This method has been widely applied to protein and DNA electron transfer reactions.
However, the current method is low in quantum yield and makes the data analysis difficult. The goal for this research is to increase the yield for the flash-quench product. Alternating the excited state electron density distribution can perturb the efficiency of electron transfer. Three ruthenium compounds were synthesized;
[Ru(bpy)2(im)2]2+, [Ru((CH3)2bpy)2(im)2]2+, and [Ru((COO−)2bpy)2(im)2]2−. For [Ru((CH3)2bpy)2(im)2]2+, the excited state quenching yield is the smallest but the formation yield of [RuIII(LL)2(im)2]3+ species is the largest. [Ru((COO−)2bpy)2(im)2]2−
has the opposite result. Driving force effect can not explain the trend of excited state quenching yield. Charge interaction and hydrophobic interaction play important roles in these results. Corresponding ruthenium carbonate complex is readily to react with the surface histidine of cytochrome c to produce the ruthenium modified cytochrome c. By using the oxidative quencher, Rua63+, intramolecular electron transfer was monitored by transient absorption spectra. Similar to the model compound,
Ru((CH3)2bpy)2(im)(His33)-Fe2+-cyt c has the largest quantum yield of intramolecular electron transfer and the smallest for Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c.
Although driving force favors for Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c, cage effect and chemical reaction on the methyl substitutent are other variable factors in the trend.
Introduction
Long range electron transfer is extremely important in biological system such as photosynthesis and cellular respiration. The photosynthesis of plants is by far the most important energy generation process in nature. To convert the transient energy of photon into stable chemical energy, three integrated membrane protein complexes involved are as following: photosystem II (PSII), cytochrome b6f (cyt b6f), and photosystem I (PSI).1-3 Electron transfer takes place between these proteins which is known as the light reaction. In the light reaction, light is converted to the energy in the form of proton gradient and NADPH as the following equation:
V 14 . 1 E 2 O H 1 NADPH photons
4 NADP O
H
2
2 o
(1)Equation 1 is a concise final overall reaction, but there are numerous electron transfer steps involved. Some steps are just electron transfer and others are proton coupled electron transfer. The electron transport chain in photosynthetic reaction contains sequential electron transfer reactions, step-by-step over a long distance (Figure 1).
2H+
Figure 1. Photosynthetic electron transport chain. OEC: oxygen evolving complex;
TyrZ: tyrosine; P680: reaction center chlorophyll; Pheo: pheophytin; QA and QB: bound plastoquinone; PQH2: protonated and reduced plastoquinone; FeS: Rieske iron-sulfur center; PC: plastocyanin; P700: reaction center chlorophyll; A0:
chlorophyll; A1: phylloquinone; FX and FA/FB: iron-sulfur center; Fd: ferredoxin; FNR:
ferredoxin/NADP+ reductase. NADPH: nicotinamide adenine dinucleotide phosphate.
ADP and ATP: adenosine diphosphate and adenosine triphosphate.
Assuming that the absorbed wavelength is at 700 nm (~40 kcal/mol), four photons absorbed will provide 160 kcal/mol as in equation 1. However, after long range electron transfer, the energy storage is about 50 kcal/mol at most. There are many charge separation states lie in as the intermediates and it consumes some energy for each step. Therefore, only about one-third of the energy is stored. Nonetheless photosynthesis of plants is still the most efficiently species to store the solar energy by far. Solar energy is the handiest, cleanest, and most economical energy on earth.
Simulating the mechanism of photosynthesis of plants by artificial photosynthesis
reaction center and enhancing the reaction yield are the goals for many scientists nowadays.4-6
Cellular respiration supplies energy to all the activities of the living beings. In the beginning, glucose is converted to pyruvate in the process of glycolysis. Pyruvates undergo oxidative decarboxylation and enter the citric acid cycle. NADH is the product from citric acid cycle and it initializes the electron transfer to promote adenosine triphosphate (ATP) production. The electron transport chain in cellular respiration is shown in Figure 2. Four membrane-bound protein complexes involve and three of them are proton pumps.7-9 Again, many electron transfer steps take place among the protein complexes. Finally, oxygen is reduced to water molecule and ATP is formed to provide chemical energy within cells for metabolism. Step-by-step electron transfer is crucial for the long range electron transfer. For instance, the distance between reaction center of cytochrome c (cyt c) and cytochrome c oxidase is above 20 Å .10 How to efficiently transfer electron without wasting unnecessary energy across a long distance in the living system has aroused many researchers’
attention.
NAD++ H2O
Figure 2. Electron transport chain of cellular respiration. Complex I: NADH
dehydrogenase; Complex II: succinate dehydrogenase; Complex III: cytochrome bc1
complex; Complex IV: cytochrome c oxidase; FMN: flavin mononucleotide; FeS:
iron-sulfur cluster; FAD: flavin adenine dinucleotide; CoQ: coenzyme Q, ubiquinone.
Metalloproteins function as electron transport are usually small (molecular weight range from 10000~15000) and water soluble. Cyt c, a heme protein, is a typical electron transport protein and plays an important role in biological system.11 The iron in cyt c has two oxidation states; Fe2+ and Fe3+, both show distinct
UV-Visible spectra which give a perfect spectroscopic handle for studying the redox chemistry of cyt c.
Ruthenium bipyridine type complex has been utilized to understand the long range electron transfer in proteins.12-18 It is soluble in aqueous and organic solvent.
Therefore, it is ready to apply to biological system. Ruthenium bipyridine type complex is the famous photosensitizer and has been investigated extensively.
Ruthenium trisbipyridine, [Ru(bpy)3]2+, has a metal-to-ligand charge-transfer (MLCT) absorption band in the visible region. Electronically excited [Ru(bpy)3]2+ has a typical
3MLCT emission profile in the visible region and the excited state energy (E00) of [Ru(bpy)3]2+ is 2.10 eV. In the ground state, [Ru(bpy)3]2+ is a stable compound which requires -1.26 V to reduce or oxidize (Figure 3). However, at the excited state,
*[Ru(bpy)3]2+ is highly reactive. With proper redox partner presents, *[Ru(bpy)3]2+
behaves as a strong reductant or oxidant.19 In addition, the excited state lifetime of [Ru(bpy)3]2+ is about 600 ns which is long enough for photoinduced bimolecular reaction.
Figure 3. Modified Latimer diagram of [Ru(bpy)3]2+ complex (the present potential are standard reduction potential versus SCE).
There are several methods to study electron transfer in ruthenium modified cytochrome c. Millett and Durham had singly labeled ruthenium bis(bipyridine) dicarboxybipyridine, [Ru(bpy)2(dcbpy)]2+, at lysine amino group on cyt c.20 Upon exciting to MLCT excited state, [Ru(bpy)2(dcbpy)]2+ behaves similar to that of well-known [Ru(bpy)3]2+ complex. With Fe3+-cyt c attached, *[Ru(bpy)2(dcbpy)]2+
acts as a strong reducing agent and transfers electron to Fe3+-cyt c due to the large
driving force (photoinduced reaction). The resulting product is metastable state and undergoes the thermodynamic favored electron recombination to the ground state.
Detail mechanism is outlined in Scheme 1.
Scheme 1. Mechanism of the direct photoinduced electron transfer for
Ru2+(bpy)2(dcbpy)-Lys-Fe3+-cyt c (Ru2+
Fe
3+). Where kd is the excited state decay rate constant, kFET is the forward electron transfer rate constant and kBET is the back electron transfer rate constant.kFET
The above method is called direct photoinduced electron transfer which has been widely adopted to study electron transfer in many fields. However, the types of electron transfer reaction are limited by the excited state lifetime of the
photosensitizer. If the electron transfer reaction is slower than the excited state decay, the method would fail. In the biological system, the distance between electron donor and acceptor is quite long so that the electron transfer reaction is generally slow.
Therefore, direct photoinduced electron transfer by ruthenium bipyridine type complexes is inadequate for probing long range electron transfer.
Flash-quench method is developed to take the advantage of the robust excited state of ruthenium bipyridine type complexes. The lifetime of reactive species is dramatically prolonged. Chang and Gray had modified [Ru(bpy)2(im)]2+ at histidine amino group on cyt c surface.21 Histidine has an imidazole side chain which may serve as a ligand. Therefore, mixing coordinatively unsaturated ruthenium complex, [Ru(bpy)2(H2O)2]2+, and cyt c and adding excess imidazole to replace remaining aqua ligand result ruthenium modified protein, Ru(bpy)2(im)(His33)-cyt c. This protein modification has hence been widely adopted to explore the long range electron transfer in biomolecules. Although neither [Ru(bpy)2(im)(His33)]2+ nor cyt c has long-lived excited state, the electron transfer reaction is initiated and monitored according to Scheme 2.
Scheme 2. Mechanism of the flash-quench method for Ru2+(bpy)2(im)(His33)-Fe2+-cyt c (Ru2+
Fe
2+). Where kd is the excited state decay rate constant, k1 is the quenching rate constant, kET is the electron transfer rate constant, k2 is the intermolecular charge recombination rate constant and Q is the oxidative quencher.h kd
In contrast to the direct photoinduced electron transfer, an oxidative quencher (Q) has been added. Photoinduced bimolecular reaction between the ruthenium excited state and oxidative quencher produces a strong oxidant, Ru3+. The consequent electron transfer between Ru3+ and Fe2+-cyt c proceeds. Even though the lifetime of ruthenium excited state is short, high concentration of oxidative quencher is sufficient to produce partial Ru3+ rapidly. The Ru3+ has much longer lifetime (~100 ms) compared to the original excited state (60 ns). At this stage, time window is much wider to study most of long range electron transfer reactions. On the other hand, back reaction between Ru3+ and reduced quencher, Q, is very slow (in seconds) due to the low concentration of both Ru3+ and Q.
Flash-quench method is powerful for extending the lifetime of a strong
oxidant/reductant. However, it also has a disadvantage which is low quantum yield for generating this long-lived oxidant/reductant. The quantum yield is related to the excited state quenching yield which depends on the quenching rate constant, the excited state lifetime of photosensitizer and the concentration of quencher (means it is concentration dependent). However the variable factors of the quenching rate constant are extensive and the added concentration of quencher is limited. Therefore, to
increase the formation yield of Ru3+, prolonging the excited state lifetime is necessary.
In this chapter, 2,2’-bipyridine (bpy) ligand and its substituent, such as
4,4’-dimethyl-2,2’-bipyridine (dm) and 4,4’-dicarboxyl-2,2’-bipyridine (dc) have been coordinated with ruthenium to form the complexes of [Ru(LL)2(im)2]2+ (LL = bpy, dm and dc; im = imidazole). Three ruthenium modified cytochrome c are synthesized. The effect of electron donating and withdrawing substituents on the bipyridine ligand to the bimolecular quenching reaction is discussed.
Experimental Section
Materials
Imidazole (im), Na2HPO4, NaH2PO4·H2O and NaCl were purchased from Merck.
Horse heart cytochrome c (cyt c) and Na2S2O4 were obtained form Aldrich. Ligands of 2,2’-bipyridine (bpy) was obtained from Fluka and 1,10-phenanthroline (phen) was purchased from Alfa Aesar. Ru(NH3)6Cl3 was purchased from Strem. All chemicals were used as received. Milli-Q-grade water (18.3 M·cm) was used to prepare all aqueous solution. Organic solvents for the synthesis reaction were reagent grade.