Electron transfer from Cyt b 559 and tyrosine-D to the S2 and S3 states of the water oxidizing complex in photosystem II at cryogenic temperatures

The Mn₄CaO₅ cluster of photosystem II (PSII) catalyzes the oxidation of water to molecular oxygen through the light-driven redox S-cycle. The water oxidizing complex (WOC) forms a triad with Tyrosine_Z and P₆₈₀, which mediates electrons from water towards the acceptor side of PSII. Under certain conditions two other redox-active components, Tyrosine_D (Y_D) and Cytochrome b ₅₅₉ (Cyt b ₅₅₉) can also interact with the S-states. In the present work we investigate the electron transfer from Cyt b ₅₅₉ and Y_D to the S₂ and S₃ states at 195 K. First, Y_D ^? and Cyt b ₅₅₉ were chemically reduced. The S₂ and S₃ states were then achieved by application of one or two laser flashes, respectively, on samples stabilized in the S₁ state. EPR signals of the WOC (the S₂-state multiline signal, ML-S₂), Y_D ^? and oxidized Cyt b ₅₅₉ were simultaneously detected during a prolonged dark incubation at 195 K. During 163 days of incubation a large fraction of the S₂ population decayed to S₁ in the S₂ samples by following a single exponential decay. Differently, S₃ samples showed an initial increase in the ML-S₂ intensity (due to S₃ to S₂ conversion) and a subsequent slow decay due to S₂ to S₁ conversion. In both cases, only a minor oxidation of Y_D was observed. In contrast, the signal intensity of the oxidized Cyt b ₅₅₉ showed a two-fold increase in both the S₂ and S₃ samples. The electron donation from Cyt b ₅₅₉ was much more efficient to the S₂ state than to the S₃ state.     

Electron transfer in photosystem II at cryogenic temperatures

The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*)

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.     

Ammonia displaces methanol bound to the water oxidizing complex of photosystem II in the S2 state

Ammonia and methanol both bind to the water oxidising complex of photosystem II during its turnover, possibly at sites where water binds during the normal water oxidation process. We have investigated the interaction between these two water analogues at the S2 state of the water oxidising cycle using electron magnetic resonance techniques. We find evidence that ammonia displaces methanol from its binding site.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and YZ radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S₁ and S₃ states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11–15 signal, were detected in Ca²⁺-depleted PS II. The g=11–15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S₂ in Ca²⁺-depleted PS II. The singlet-like signal was associated with the g=11–15 signal but not with the Y_Z (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y_Z radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y_Z radical was discussed.     

Two-step mechanism of photodamage to photosystem II: step 1 occurs at the oxygen-evolving complex and step 2 occurs at the photochemical reaction center

Under strong light, photosystem II (PSII) of oxygenic photosynthetic organisms is inactivated, and this phenomenon is called photoinhibition. In a widely accepted model, photoinhibition is induced by excess light energy, which is absorbed by chlorophyll but not utilized in photosynthesis. Using monochromatic light from the Okazaki Large Spectrograph and thylakoid membranes from Thermosynechococcus elongatus, we observed that UV and blue light inactivated the oxygen-evolving complex much faster than the photochemical reaction center of PSII. These observations suggested that the light-induced damage was associated with a UV- and blue light-absorbing center in the oxygen-evolving complex of PSII. The action spectrum of the primary event in photodamage to PSII revealed the strong effects of UV and blue light and differed considerably from the absorption spectra of chlorophyll and thylakoid membranes. By contrast to the photoinduced inactivation of the oxygen-evolving complex in untreated thylakoid membranes, red light efficiently induced inactivation of the PSII reaction center in Tris-treated thylakoid membranes, and the action spectrum resembled the absorption spectrum of chlorophyll. Our observations suggest that photodamage to PSII occurs in two steps. Step 1 is the light-induced inactivation of the oxygen-evolving complex. Step 2, occurring after step 1 is complete, is the inactivation of the PSII reaction center by light absorbed by chlorophyll. We confirmed our model by illumination of untreated thylakoid membranes with blue and UV light, which inactivated the oxygen-evolving complex, and then with red light, which inactivated the photochemical reaction center.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

Decay products of the S(3) state of the oxygen-evolving complex of photosystem II at cryogenic temperatures. Pathways to the formation of the S = 7/2 S(2) state configuration

The water-oxidizing complex of photosystem II cycles through five oxidation states, denoted S(i)() (i = 0-4), during water oxidation to molecular oxygen, which appears at the (transient) S(4) state. The recent detection of bimodal EPR signals from the S(3) state [Matsukawa, T., Mino, H., Yoneda, D., Kawamori, A. (1999) Biochemistry 38, 4072-4077] has drawn significant attention to this critical state. An interesting property of the S(3) state is the sensitivity to near-IR (NIR) light excitation. Excitation of the S(3) state by near-IR light at cryogenic temperatures induces among other signals a derivative-shaped EPR signal at g= 5 [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. The signal bears unexpected similarities to a signal observed earlier in samples that had undergone multiple turnovers and subsequently had been stored at 77 K for a week or longer [Nugent, J. H. A., Turconi, S., and Evans, M. C. W. (1997) Biochemistry 36, 7086-7096]. Recently, both signals were assigned to an S = 7/2 configuration of the Mn cluster [Sanakis, Y., Ioannidis, N., Sioros, G., and Petrouleas, V. (2001) J. Am. Chem. Soc. 123, 10766-10767]. In the present study, we employ bimodal EPR spectroscopy to investigate the pathways of formation of this unusual state. The following observations are made: (i) The g = 5 signal evolves in apparent correlation with the diminution of the S(3) state signals during the slow (tens of hours to several days range) charge recombination of S(3) with Q(A)(-) at 77 K. The tyrosyl radical D* competes with S(3) for recombination with Q(A)(-), the functional redox couple at cryogenic temperatures inferred to be D*/D(-). Transfer to -50 degrees C and above results in the relaxation of the g = 5 to the multiline and g = 4.1 signals of the normal S(2) state. (ii) The transition of S(3) to the state responsible for the g = 5 signal can be reversed by visible light illumination directly at -30 degrees C or by illumination at 4.2 K followed by brief (2 min) transfer to -50 degrees C in the dark. The latter step is required in order to overcome an apparent thermal activation barrier (charge recombination appears to be faster than forward electron transfer at 4.2 K). (iii) The "g = 5" state can be reached in a few tens of minutes at 4.2 K by near-IR light excitation of the S(3) state. This effect is attributed to the transfer of the positive hole from the Mn cluster to a radical (probably tyr Z), which recombines much faster than the Mn cluster with Q(A)(-). (iv) The above properties strongly support the assignment of the configuration responsible for the g = 5 signal to a modified S(2) state, denoted S(2)'. Evidence supporting the assignment of the S(2)' to a proton-deficient S(2) configuration is provided by the observation that the spectrum of S(2) at pH 8.1 (obtained by illumination of the S(1) state at -30 degrees C) contains a g = 5 contribution.     

Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures

Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects of triacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at -15 degrees C or liquid nitrogen temperatures. We used differential scanning calorimetry (DSC) to monitor enthalpy and temperature of water and TAG phase transitions within spores of five fern species: Pteris vittata, Thelypteris palustris, Dryopteris filix-mas, Polystichum aculeatum, Polystichum setiferum. The analyses suggested that these fern spores contained between 26% and 39% TAG, and were comprised of mostly oleic (P. vittata) or linoleic acid (other species) depending on species. The water contents at which water melting events were first observable ranged from 0.06 (P. vittata) to 0.12 (P. setiferum)gH(2)Og(-1)dry weight, and were highly correlated with water affinity parameters. In spores containing more than 0.09 (P. vittata) to 0.25 (P. setiferum)gH(2)Og(-1)dry weight, some water partitioned into a near pure water fraction that melted at about 0 degrees C. These sharp peaks near 0 degrees C were associated with lethal freezing treatments. The enthalpy of water melting transitions was similar in fern spores, pollen and seeds; however, the unfrozen water content was much lower in fern spores compared to other forms of germplasm. Though there is a narrow range of water contents appropriate for low temperature storage of fern spores, water content can be precisely manipulated to avoid both desiccation and freezing damage.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and Y(Z) radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S1 and S3 states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11-15 signal, were detected in Ca2+-depleted PS II. The g=11-15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S2 in Ca2+-depleted PS II. The singlet-like signal was associated with the g=11-15 signal but not with the Y(Z) (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y(Z) radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y(Z) radical was discussed.     

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.     

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.     

Modulation of flash-induced photosystem II fluorescence by events occurring at the water oxidizing complex.

The mechanism of flash-induced changes with a periodicity of four in photosystem II (PSII) fluorescence was investigated with the aim of further using fluorescence measurements as an approach to studying the structural and functional organization of the water-oxidizing complex (WOC). The decay of the flash-induced high fluorescence state of PSII was measured with pulse amplitude modulated fluorometry in thylakoids and PSII enriched membrane fragments. Calculated QA- decay was well described by three exponential decay components, reflecting QA- reoxidation with halftimes of 450 and 860 micros, 2 and 7.6 ms, and 111 and 135 ms in thylakoids and PSII membranes, respectively. The effect of modification of the PSII donor side by changing pH or by removal of the extrinsic 17 and 24 kDa proteins on period four oscillations in both maximum fluorescence yield and the relative contribution of QA- reoxidation reactions was compared to flash-induced oxygen yield. The four-step oxidation of the manganese cluster of the WOC was found to be necessary but not sufficient to produce modulation of PSII fluorescence. The capacity of the WOC to generate molecular oxygen was also required to observe a period four in the fluorescence; however, direct quenching by oxygen was not responsible for the modulation. Potential mechanisms responsible for the periodicity of four in both maximum fluorescence yield pattern and flash-dependent changes in proportion of centers with different QA- reoxidation rates are discussed with respect to intrinsic deprotonation events occurring at the WOC.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

Electron transfer between the two photosystems. I. Flash excitation under oxidizing conditions

The redox changes of cytochrome b-563 (cytochrome b), cytochrome f, plastocyanin and P-700 were measured on dark-adapted chloroplasts after illumination by a series of flashes in oxidizing conditions (0.1 mM ferricyanide). In these conditions, the plastoquinone pool is fully oxidized and the only available plastoquinol are those formed by Photosystem (PS) II reaction. According to the two-electron gate mechanism proposed by Bouges-Bocquet (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250–256), plastoquinol is mainly formed after the second and the fourth flashes. After the second flash, the reoxidation of plastoquinol occurs by a concerted reaction which reduces most of the cytochrome b present and a fraction of PS I donors. Most of these electrons are stored on P-700, which implies a large equilibrium constant between the secondary PS I donors and P-700. One electron is stored on cytochrome b during a time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{s}$) much longer than the dark interval between flashes. After the fourth flash, a new plastoquinol molecule is formed, which induces the reduction of PS I donors with no corresponding further reduction of cytochrome b. The number of electrons transferred after the fourth flash is larger than that transferred after the second flash although the rate of transfer is lower. To interpret these data, we assume that the plastoquinol formed after the fourth flash is reoxidized by a second concerted reaction: one electron is directly transferred to PS I donors while the other cooperates with the electron stored on cytochrome b to reduce a plastoquinone molecule localized on a site close to the outer face of the membrane. This newly formed plastoquinol crosses the membrane and transfers a second electron to PS I donors. This interpretation resembles a model proposed by Velthuys (Velthuys, B.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2765?2769) and which belongs to the modified Q-cycle class of models.     

Novel Ist1-Did2 complex functions at a late step in multivesicular body sorting

In Saccharomyces cerevisiae, integral plasma membrane proteins destined for degradation and certain vacuolar membrane proteins are sorted into the lumen of the vacuole via the multivesicular body (MVB) sorting pathway, which depends on the sequential action of three endosomal sorting complexes required for transport. Here, we report the characterization of a new positive modulator of MVB sorting, Ist1. We show that endosomal recruitment of Ist1 depends on ESCRT-III. Deletion of IST1 alone does not cause cargo-sorting defects. However, synthetic genetic analysis of double mutants of IST1 and positive modulators of MVB sorting showed that ist1Delta is synthetic with vta1Delta and vps60Delta, indicating that Ist1 is also a positive component of the MVB-sorting pathway. Moreover, this approach revealed that Ist1-Did2 and Vta1-Vps60 compose two functional units. Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and, like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provide evidence that the ist1Delta mutation exhibits a synthetic interaction with mutations in VPS2 (DID4) that compromise the Vps2-Vps4 interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60 complexes independently modulate late steps in the MVB-sorting pathway.     

Bidirectional electron transfer in photosystem I: replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone A1B with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures

A conserved tryptophan residue located between the A(1B) and F(X) redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A(1A) and the A(1B) phyllo(semi)quinones. The kinetics of A(1)(-) reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F(X) is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F(X) leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F(A) and F(B) by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A(1B).     

Electron transfer from cytochromeb 5 to cytochromec

The reaction of cytochromeb ₅ with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.     

Electron transfer from cytochrome c to cupredoxins

Electron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (ΔG ⁰) and the reorganization energy. Here, we characterized ET from Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA) and its designed mutants to cupredoxins, Silene pratensis plastocyanin (PC) and Acidithiobacillus ferrooxidans rusticyanin (RC), through measurement of pseudo-first-order ET rate constants (k _obs). The influence of the ΔG ⁰ value for ET from PA to PC or RC on the k _obs value was examined using a series of designed PA proteins exhibiting a variety of E _m values, which afford the ΔG ⁰ variation range of 58–399 meV. The plots of the k _obs values obtained against the ΔG ⁰ values for both PA–PC and PA–RC redox pairs could be fitted well with a single Marcus equation. We have shown that the ET activity of cytochrome c can be controlled by tuning the E _m value of the protein through the substitution of amino acid residues located in hydrophobic-core regions relatively far from the redox center. These findings provide novel insights into the molecular design of cytochrome c, which could be utilized for controlling its ET activity by means of protein engineering. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.