EPR kinetic studies of the S−1 state in spinach thylakoids

AbstractThe YZ decay kinetics in a formal S−1 state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S−1 state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S−1 state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the YZ re-reduction rate by the S−1 state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S−1 to S0 transition, which is in the order of the S3 to S0 transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S−1 to S0 transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples

Starch, Lipid, and Protein Accumulation in Nutrient-Stressed Microalgal Cells Studied Using Fourier Transform Infrared Microscopy

Microalgae are fast growing organisms that can be used as feedstock for the production of biofuels. The metabolism of microalgae can be manipulated by exposing them to different environmental conditions for favoring the accumulation of lipids, carbohydrates or proteins. For example, a change in growth conditions can cause the accumulation of large amounts of lipids, representing an opportunity for biodiesel production. Monitoring changes in the composition of microalgal cells is therefore important in assessing new growth conditions. However, at present, most techniques are time consuming, invasive and expensive. Here we have used FTIR microscopy to quantify lipid, protein, and starch accumulation in Neochloris minuta cells grown in the presence and absence of nitrogen. Under nitrogen deprivation the cellular lipid composition increases by a factor of 2.4, the cellular protein concentration decreases by ~60% while the starch concentration is unaltered. These estimates of biochemical composition were derived using a variety of analytical methods, and form the basis for establishing to what extent FTIR microscopy can be used as a probe of cellular biochemical composition. We find that the distribution of materials in Neochloris minuta cells estimated directly from the FTIR spectra compare favorably with that estimated using these other analytical methods. FTIR spectroscopy is shown to be a versatile and easy-to-use tool for estimating distributions of biological materials in microalgal cells

Infrared spectroscopic identification of the C–O stretching vibration associated with the tyrosyl Z⋅ and D⋅ radicals in photosystem II2Supported by NIH GM 43272 (B.A.B.), NSF MCB 94-18164 (B.A.B.), a graduate minority supplement to NIH GM 43273 (I.A.), a graduate fellowship from Committee on Institutional Cooperation, University of Minnesota (I.A.), and a summer research fellowship from Dupont, Central Research and Development, administered through the University of Minnesota (E.T.G.).2

AbstractPhotosystem II (PSII) is a multisubunit complex, which catalyzes the photo-induced oxidation of water and reduction of plastoquinone. Difference Fourier-transform infrared (FT-IR) spectroscopy can be used to obtain information about the structural changes accompanying oxidation of the redox-active tyrosines, D and Z, in PSII. The focus of our work is the assignment of the 1478 cm−1 vibration, which is observable in difference infrared spectra associated with these tyrosyl radicals. The first set of FT-IR experiments is performed with continuous illumination. Use of cyanobacterial strains, in which isotopomers of tyrosine have been incorporated, supports the assignment of a positive 1478/1477 cm−1 mode to the C–O stretching vibration of the tyrosyl radicals. In negative controls, the intensity of this spectral feature decreases. The negative controls involve the use of inhibitors or site-directed mutants, in which the oxidation of Z or D is eliminated, respectively. The assignment of the 1478/1477 cm−1 vibrational mode is also based on control EPR and fluorescence measurements, which demonstrate that no detectable Fe2+QA− signal is generated under FT-IR experimental conditions. Additionally, the difference infrared spectrum, associated with formation of the S2QA− state, argues against the assignment of the positive 1478 cm−1 line to the C–O vibration of QA−. In the second set of FT-IR experiments, single turnover flashes are employed, and infrared difference spectra are recorded as a function of time after photoexcitation. Comparison to kinetic transients generated in control EPR experiments shows that the decay of the 1477 cm−1 line precisely parallels the decay of the D⋅ EPR signal. Taken together, these two experimental approaches strongly support the assignment of a component of the 1478/1477 cm−1 vibrational lines to the C–O stretching modes of tyrosyl radicals in PSII. Possible reasons for the apparently contradictory results of Hienerwadel et al. (Biochemistry 35 (1996) 15447–15460 and Biochemistry 36 (1997) 14705–14711) are discussed

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

AbstractThe active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5 cluster and TyrZ, a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7Å away from the Mn4CaO5 cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I− for Cl− in the cyanobacterium Thermosynechococcus elongatus with either Ca2+ or Sr2+ biosynthetically assembled into the Mn4 cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV–visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl−/I− exchange considerably slowed down the two S3TyrZ•→(S3TyrZ•)′→S0 reactions in which the fast phase, S3TyrZ•→(S3TyrZ•)′, reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on TyrZ• and the slow phase corresponds to the S0 and O2 formations and to a second proton release. The t1/2 for S0 formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the TyrZ• reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca2+ binding site and the Cl− binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl− and Ca2+ binding sites by which proton release may be channelled

Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes

Photobiocatalysts are constituted by a semiconductor with or without a light harvester that activates an enzyme. A logical source of inspiration for the development of photobiocatalysts has been natural photosynthetic centers. In photobiocatalysis, the coupling of the semiconductor and the enzyme frequently requires a natural cofactor and a relay transferring charge carriers from the semiconductor. The most widely studied photobiocatalysts so far make use of conduction band electrons of excited semiconductors to promote enzymatic reductions mediated by NAD(+)/NADH and an electron relay. The present review presents the state of the art in the field and has been organized based on the semiconductor and the reaction type including oxidations, hydrogen generation, and CO2 reduction. The possibility of direct enzyme activation by the semiconductor and the influence of the nature of mediator are also discussed as well as the use of mimics of the enzyme active center in combination with the semiconductor. The final section summarizes the state of the art of photobiocatalysis and comments on our view on future developments of the field.Financial support by the Spanish Ministry of Economy and Competitiveness (Severo Ochoa and CTQ2012-32315) is gratefully acknowledged. J.A.M.-A. acknowledges the assistance of the CSIC for the award of a Postdoctoral JAE-Doc contract co-financed by the European Social Fund.Maciá Agulló, JA.; Corma Canós, A.; García Gómez, H. (2015). Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes. Chemistry - A European Journal. 21(31):10940-10959. https://doi.org/10.1002/chem.201406437S1094010959213

On-column refolding of recombinant human interleukin-4 from inclusion bodies

Interleukin-4 (IL4) is a multifunctional cytokine which plays a key role in the immune system. Several antagonists/agonists of IL4 are reported through mutagenesis studies, but their solution structural studies using nuclear magnetic resonance (NMR) spectroscopy are hindered as milligram quantities of isotopically labeled protein are required for structural refinements. In this work, a His-tagged recombinant form of human IL4 was overexpressed in Escherichia coli under the control of a T7 promoter. The resulting inclusion bodies were separated from cellular debris by centrifugation and solubilized by 6M guanidine-HCl in the presence of reducing agents. The denatured IL4 was immobilized on Ni2+-fractogel beads and refolded in a single chromatographic step by gradual removal of denaturant. This protocol yielded 15-20mg of isotope-enriched protein from 1L of culture grown in minimal medium. The refolded protein was highly pure and was correctly folded as judged by its two-dimensional NMR spectrum. To show the successful application of this refolding protocol to IL4 variants,15N-labeled Y124D-IL4 was also prepared and its first two-dimensional NMR spectrum was presented

Photochemistry of Free and Bound Zn-Chlorophyll Analogues to Synthetic Peptides Depend on the Quinone and pH

A synthetic peptide was used as a scaffold to bind Zn-Chlorophyll (ZnChl) analogues through histidine ligation to study their photochemistry in the presence of different type of quinones. The Chl analogues were chlorin e6 (Ce6), chlorin e6 trimethyl ester, pyropheophorbide a, and pheophorbide a while the quinones were PPBQ, DMBQ, NPHQ, DBTQ, DCBQ and PBQ. The binding of each ZnChl analogue to the peptide was verified by native gel electrophoresis. First the photo-stability of the ZnChl analogues were tested under continuous light. The ZnCe6 and ZnCe6TM analogues showed the least stability judged by the loss of optical signal intensity at their Qy band. The photoactivity of each ZnChl analogue was measured in the presence of each of the six quinones using time-resolved EPR spectroscopy. DMBQ was found to be the most efficient electron acceptor when all four ZnChl analogues were compared. The light-induced electron transfer between the ZnChl analogues complexed with the peptide and DMBQ were also measured using time-resolved EPR spectroscopy. The ZnCe6–peptide complex exhibited the highest photoactivity. The electron transfer in the complex was faster and the photoactivity yield was higher than those values obtained for free ZnCe6 and DMBQ. The fast phase of kinetics can be attributed to intra-protein electron transfer in the complex since it was not observed in the presence of DMBQ–glutathione adduct. Unlike free ZnCe6, the ZnCe6–peptide complex was robust and demonstrated very similar photoactivity efficiency in pH values 10, 8.0 and 5.0. The electron transfer kinetics were pH dependent and appeared to be modulated by the peptide charge and possibly fold. The charge recombination rate was slowed by an order of magnitude when the pH value was changed from 10.0 to 5.0. The implications of constructing the photoactive peptide complexes in terms of artificial photosynthesis are discussed