Joseph T. Warden

Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

Abstract The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S 2− →S 0 conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.

Is phylloquinone an obligate electron carrier in photosystem I

Comparative quantitative analysis of phylloquinone content and photochemically competent P‐700 has been performed on photosystem I particles subjected to photolysis with ultraviolet irradiation. Nonirradiated control particles exhibit a phylloquinone/P‐700 stoichiometry of 1.9 ± 0.2. Photolysis of the photosystem I particles induces a progressive depletion of phylloquinone, however, photochemistry as assayed at room temperature by the photooxidation of P‐700 is unaffected. These data are not consistent with the assignment of phylloquinone as a functional intermediate at room temperature between P‐700 and the iron‐sulfur clusters, center A and center B.

Kinetics of the fluorescence yield of chlorophyll a2 in spinach chloroplasts at liquid nitrogen temperature du ring and following a 16 μs flash

Abstract If, at liquid nitrogen temperature, the initial fluorescence yield of chlorophyll a 2 is high ( e.g. after preillumination), a 16 μs flash produces in a few microseconds a decrease in fluorescence yield, followed by an increase, which occurs after roughly 10–20 μs, when the intensity of the flash has become negligible. It is concluded that during a flash, a quencher or quenching state T is formed, which disappears in a dark reaction in a time of the order of 10 μs. The kinetics are the same and can be interpreted in the same way as the kinetics at room temperature earlier reported by Duysens et al. (Abstr. VI. Int. Congr. on Photobiol. Bochum 1972, No. 277). If the flash is given when the initial fluorescence yield is low, then the fluorescence yield increases only markedly at the end of the flash, when the intensity has become low. Even for a strong flash, the increase is only about 20% of the maximum increase attained after a large number of flashes. This indicates that at low temperature, in contradistinction to room temperature, the reduction of the primary oxidant Q is less efficient than the formation of the quencher T. For the interpretation of the experiments it was not necessary to introduce other light-induced quenchers than T, such as the oxidized primary reductant, P + .

Investigation of the ammonium chloride and ammonium acetate inhibition of oxygen evolution by Photosystem II

Using EPR and EXAFS spectroscopies we show that high concentrations of ammonium cations at alkaline pH are required for (1) inhibition of oxygen evolution: (2) an alteration of the EPR properties of the oxygen evolving complex: (3) the ability to detect YZ; and (4) the slow reduction of the Mn complex leading to the appearance of EPR detectable Mn2+. The inhibition of S state cycling, slowing of YZ reduction, appearance of Mn2+ and the yield of a Hpp < 10 mT S3 type EPR signal are decreased by calcium addition. This indicates that these effects were probably associated with calcium depletion arising from the high concentration of ammonium cation. The ammonia-induced changes to the S2 multiline EPR signal are not affected by calcium addition. The appearance of Mn2+ is shown to be reversible on illumination, suggesting that the Mn reduced from the native state is located at or near the native site. Simulations of the interaction which give rise to the S3 EPR signal are also presented and discussed. These indicate that lineshape differences occur through small changes in the exchange component of the interaction between the manganese complex and organic radical, probably through minor structural changes between the variously treated samples.

On the mechanism of linolenic acid inhibition in photosystem II

Recent studies in our laboratory have reexamined the interaction of the unsaturated fatty acid, linolenic acid, with Photosystem II and have documented two principal regions of inhibition: one associated with the donor complex (Signal 2f or D1) to the reaction center, and the other located on the reducing side between pheophytin and Qa (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271). A further characterization of fatty acid inhibition of secondary electron transport in Photosystem II at room and cryogenic temperatures is presented in this paper. These studies demonstrate that linolenic acid, and related fatty acid analogs, eliminate the transient absorption increase at 320 nm, attributed to Qa-; abolish the production, either chemically or photochemically, of the ESR signal (Q-Fe) associated with the bound quinone acceptor, Qa-; and prevent the photooxidation of Signal 2(1t)(D1) at cryogenic temperature. Linolenic-acid-treated samples are characterized by a high initial fluorescence yield (Fi) equivalent to the maximum level of fluorescence (Fmax); however, the spin-polarized triplet, associated with the reaction-center electron donor, P-680, is observed only in inhibited samples that have been prereduced with sodium dithionite. These results suggest the presence of an additional acceptor intermediate between pheophytin and Qa. The donor-assisted photoaccumulation of pheophytin anion in Photosystem II particles, as monitored by the decline of fluorescence yield, is inhibited by linolenic acid. Redox titrations of the fluorescence yield in control and inhibited preparations demonstrate that the midpoint potential for the primary acceptor for Photosystem II is insensitive to the fatty acid (Em approximately -583 mV) and thus indicate that primary photochemistry is functional during linolenic-acid inhibition. These data are consistent with the hypothesis that unsaturated fatty acids inhibit secondary electron transport in Photosystem II via displacement of endogenous quinone from quinone-binding peptides.

Interaction of linolenic acid with bound quinone molecules in photosystem II: time-resolved optical and electron spin resonance studies

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.

Student reactions and learning: evaluation of a biochemistry course that uses web technology and student collaboration

Many academic institutions across the country are incorporating computer and Internet technology into their classrooms. Rensselaer has developed a studio classroom that incorporates student collaboration and Internet technology. The purpose of this research was to examine student reactions and learning in an undergraduate biochemistry course taught in a studio classroom. Student reactions to the course, the technology, and working in groups were positive. Students liked using the technology, felt it helped them to learn the material, and thought working in groups was beneficial for learning. Pre‐test and post‐test grades on a standardized examination indicated a significant increase in learning. In addition, positive reactions to using the technology in the course, as well as student confidence to do well in the course, were significantly related to improved course performance.

Redox study of electron donation to P-680 in Photosystem II

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680+ in both preparations showed biphasic recovery phases with half-times of 42 and 625 microseconds at pH 4.5. The magnitude of the 42 microseconds phase of P-680+ rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 +/- 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5-6.0. In contrast, the magnitude of the 625 microseconds phase of P-680+ rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with Em approximately equal to 240 mV, termed D0, serves as a rapid donor (t 1/2 less than or equal to 2 microseconds) to P-680+ in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D1, is functional only when D0 becomes oxidized.

Electron spin resonance and electrospray ionization mass spectroscopic studies of the interaction of alkali and alkaline earth cations with manganese bis(μ-oxo) dimers

Abstract The dimer [(3,3′-17-crown-6-SALPN)Mn IV (μ-O)] 2 ( 2 ) was prepared by the reaction of (3,3′-17-crown-6-SALPN)Mn(II) with O 2 in CH 3 CN. Dimer 2 when reacted with two molar equivalents of NaPF 6 , KPF 6 , CaTf 2 or BaTf 2 (Tf − is SO 3 CF 3 − formed the complexes 2 · 2NaPF 6 , 2 ·KPF 6 , 2 ·2CaTf 2 or 2 ·2BaTf 2 , respectively. The dimers were reduced to the mixed-valent Mn III ,Mn IV state either chemically with Cp 2 Co or electrochemically and their ESR spectra recorded. The ESR spectrum of each mixed-valent dimer showed a 16-line pattern characteristic of a valence trapped antiferromagnetically coupled species. The cation containing Mn IV ,Mn IV bis(μ-oxo) dimers were also analyzed by electrospray ionization mass spectrometry using CH 2 Cl 2 and CH3CN as solvents. Singly charged cations corresponding to the loss of one anion from the parent complex were observed for all four compounds. In addition, ions were detected in which cation exchange had occurred.

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I. II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters.

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77-84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with [D] = 279 X 10(-4) cm-1 and [E] = 39 X 10(-4) cm-1. The triplet resonances titrate with a midpoint potential of +380 +/- 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and delta Hpp = 1.0 mT. Deconvolution of the P-700+ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species A0, which is characterized by g = 2.0033 +/- 0.0004 and delta Hpp = 1.0 +/- 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A1 in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A1 is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A1 in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A1 from its binding site.