Inverted region in the reaction of the quinone reduction in the A1-site of photosystem I from cyanobacteria.

Photosystem I from the menB strain of Synechocystis sp. PCC 6803 containing foreign quinones in the A1 sites was used for studying the primary steps of electron transfer by pump-probe femtosecond laser spectroscopy. The free energy gap (- ΔG) of electron transfer between the reduced primary acceptor A0 and the quinones bound in the A1 site varied from 0.12 eV for the low-potential 1,2-diamino-anthraquinone to 0.88 eV for the high-potential 2,3-dichloro-1,4-naphthoquinone, compared to 0.5 eV for the native phylloquinone. It was shown that the kinetics of charge separation between the special pair chlorophyll P700 and the primary acceptor A0 was not affected by quinone substitutions, whereas the rate of A0 → A1 electron transfer was sensitive to the redox-potential of quinones: the decrease of - ΔG by 400 meV compared to the native phylloquinone resulted in a ~ fivefold slowing of the reaction The presence of the asymmetric inverted region in the ΔG dependence of the reaction rate indicates that the electron transfer in photosystem I is controlled by nuclear tunneling and should be treated in terms of quantum electron-phonon interactions. A three-mode implementation of the multiphonon model, which includes modes around 240 cm-1 (large-scale protein vibrations), 930 cm-1 (out-of-plane bending of macrocycles and protein backbone vibrations), and 1600 cm-1 (double bonds vibrations) was applied to rationalize the observed dependence. The modes with a frequency of at least 1600 cm-1 make the predominant contribution to the reorganization energy, while the contribution of the "classical" low-frequency modes is only 4%.

Critical evaluation of electron transfer kinetics in P700-FA/FB, P700-FX, and P700-A1 Photosystem I core complexes in liquid and in trehalose glass.

This work aims to fully elucidate the effects of a trehalose glassy matrix on electron transfer reactions in cyanobacterial Photosystem I (PS I). Forward and backward electron transfer rates from A1A- and A1B- to FX, and charge recombination rates from A0-, A1B-, A1A-, FX-, and [FA/FB]- to P700+ were measured in P700-FA/FB complexes, P700-FX cores, and P700-A1 cores, both in liquid and in a trehalose glassy matrix at 11% humidity. By comparing CONTIN-resolved kinetic events over 6 orders of time in increasingly simplified versions of PS I at 480 nm, a wavelength that reports primarily A1A-/A1B- oxidation, and over 9 orders of time at 830 nm, a wavelength that reports P700+ reduction and A0- oxidation, assignments could be made for nearly all of the resolved kinetic phases. Trehalose-embedded PS I samples demonstrated partially arrested forward electron transfer. The fractions of complexes in which electron transfer did not proceed beyond A0, A1 and FX were 53%, 16% and 22%, respectively, with only 10% of electrons reaching the terminal FA/FB clusters. The ~10 µs and ~150 µs components in both liquid and trehalose-embedded PS I were assigned to recombination between A1B- and P700+ and between A1A- and P700+, respectively. The kinetics and amplitudes of these resolved kinetic phases in liquid and trehalose-embedded PS I samples could be well-fitted by a kinetic model that allowed us to calculate the asymmetrical contribution of the A1A- and A1B- quinones to the electrochromic signal at 480 nm. Possible reasons for these effects are discussed.

Spectral Hole Burning in Cyanobacterial Photosystem I with P700 in Oxidized and Neutral States.

We explored the rich satellite hole structures emerging as a result of spectral hole burning in cyanobacterial photosystem I (PSI) and demonstrated that hole burning properties of PSI, particularly at high resolution, are strongly affected by the oxidation state of the primary donor P700, as P700+ effectively quenches the excitations of the lowest-energy antenna states responsible for fluorescence. Obtaining better control of this variable will be crucial for high-resolution ensemble experiments on protein energy landscapes in PSI. The separate nonphotochemical spectral hole burning (NPHB) signatures of various red antenna states were obtained, allowing for additional constraints on excitonic structure-based calculations. Preliminary evidence is presented for an additional red state of PSI of T. elongatus peaked at 712.6 nm, distinct from previously reported C708 and C715 states and possibly involving chlorophyll B15. Excitation at wavelengths as long as 800 nm results in charge separation at cryogenic temperatures in PSI also in Synechocystis sp. PCC 6803. Both the "P700+ minus P700" holes and nonphotochemical spectral holes were subjected to thermocycling. The distribution of barriers manifesting in recovery of the "P700+ minus P700" signature contains two components in sample-dependent proportions, likely reflecting the percentages of FA and FB clusters being successfully prereduced before the optical experiment. The barrier distribution for the recovery of the lower-energy nonphotochemical spectral holes resembles those observed for other pigment-protein complexes, suggesting similar structural elements are responsible for NPHB. Higher-energy components exhibit evidence of "domino effects" such as shifts of certain bands persisting past the lower-energy hole recovery. Thus, conformational changes triggered by excitation of one pigment likely can affect multiple pigments in this tightly packed system.

[2Fe-2S] proteins in Chlorosomes: redox properties of CsmI, CsmJ, and CsmX of the Chlorosome envelope of Chlorobaculum tepidum.

The chlorosome envelope of Chlorobaculum tepidum contains 10 polypeptides, three of which, CsmI, CsmJ, and CsmX, have an adrenodoxin-like domain harboring a single [2Fe-2S] cluster. Mutants that produced chlorosomes containing two, one, or none of these Fe-S proteins were constructed [Li, H., et al. (2013) Biochemistry 52, preceding paper in this issue ( DOI: 10.1021/bi301454g )]. The electron paramagnetic resonance (EPR) spectra, g values, and line widths of the Fe-S clusters in individual CsmI, CsmJ, and CsmX proteins were obtained from studies with isolated chlorosomes. The Fe-S clusters in these proteins were characterized by EPR and could be differentiated on the basis of their g values and line widths. The EPR spectrum of wild-type chlorosomes could be simulated by a 1:1 admixture of the CsmI and CsmJ spectra. No contribution of CsmX to the EPR spectrum of chlorosomes was observed because of its low abundance. In chlorosomes that contained only CsmI or CsmJ, the midpoint potential of the [2Fe-2S] clusters was -205 or 8 mV, respectively; the midpoint potential of the [2Fe-2S] cluster in CsmX was estimated to be more oxidizing than -180 mV. In wild-type chlorosomes, the midpoint potentials of the [2Fe-2S] clusters were -348 mV for CsmI and 92 mV for CsmJ. The lower potential for CsmI in the presence of CsmJ, and the higher potential for CsmJ in the presence of CsmI, were attributed to interactions that occur when these proteins form complexes in the chlorosome envelope. The redox properties of CsmI and CsmJ are consistent with their proposed participation in the transfer of electrons to and from quenchers of energy transfer in chlorosomes.

Spectral resolution of the primary electron acceptor A0 in Photosystem I.

The reduced state of the primary electron acceptor of Photosystem I, A(0), was resolved spectroscopically in its lowest energy Q(y) region for the first time without the addition of chemical reducing agents and without extensive data manipulation. To carry this out, we used the menB mutant of Synechocystis sp. PCC 6803 in which phylloquinone is replaced by plastoquinone-9 in the A(1) sites of Photosystem I. The presence of plastoquinone-9 slows electron transfer from A(0) to A(1), leading to a long-lived A(0)(-) state. This allows its spectral signature to be readily detected in a time-resolved optical pump-probe experiment. The maximum bleaching (A(0)(-) - A(0)) was found to occur at 684 nm with a corresponding extinction coefficient of 43 mM(-1) cm(-1). The data show evidence for an electrochromic shift of an accessory chlorophyll pigment, suggesting that the latter Q(y) absorption band is centered around 682 nm.

Time-resolved FTIR difference spectroscopy for the study of photosystem I particles with plastoquinone-9 occupying the A1 binding site.

In photosystem I from plants and cyanobacteria a phylloquinone molecule, called A1, functions as the secondary electron acceptor. In cyanobacteria, genes that encode for proteins involved in phylloquinone biosynthesis can be deleted. Here, we have studied three different gene deletion mutants called menB, menD, and menE mutants. In these mutants, plastoquinone-9 occupies the A1 binding site. Using time-resolved, step-scan FTIR difference spectroscopy we have produced A1(-)/A1 FTIR difference spectra for menB, menD, and menE photosystem I particles at 77 K. These difference spectra show that the P700 triplet state ((3)P700) is formed in a large fraction of the particles. Infrared spectral signatures that are not due to (3)P700 are also observed in the spectra and are suggested to be associated with plastoquinone-9 anion formation in a portion of the particles. By subtracting the known (3)P700 spectral signatures, we produce an A1(-)/A1 FTIR difference spectrum for PS I particles with plastoquinone-9 occupying the binding site. This spectrum shows that a band that we have previously assigned to a C:-O mode of the phylloquinone anion in WT A1(-)/A1 FTIR DS down-shifts approximately 8 cm(-1) when plastoquinone-9 occupies the A1 binding site. Using density functional theory type calculations to produce anion minus neutral infrared difference spectra for both phylloquinone and plastoquinone-9, it is shown that such a downshift is reasonable. A1(-)/A1 FTIR difference spectra, obtained using menB mutant photosystem I particles that were incubated in the presence of phylloquinone, are found to be very similar to those obtained using normal WT photosystem I particles. This result indicates that we were able to reincorporate phylloquinone back into the A1 binding site and that the reincorporated phylloquinone and its immediate protein environment, in both the neutral and anion state, are very similar to that found in wild type photosystem I particles. For the reconstituted menB mutant photosystem I particles, no spectral signatures associated with (3)P700 are observed, indicating that phylloquinone occupies the A1 site in all of the reconstituted menB particles.

FTIR difference spectroscopy in combination with isotope labeling for identification of the carbonyl modes of P700 and P700+ in photosystem I.

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.