The binding of the primary quinone anion radical Q(A)(-.) in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides species was investigated with electron spin echo envelope modulation (ESEEM).... Show moreThe binding of the primary quinone anion radical Q(A)(-.) in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides species was investigated with electron spin echo envelope modulation (ESEEM). The ESEEM spectra, at two microwave frequencies, of Zn-substituted RCs of Rb. sphaeroides R26 showed interactions of the unpaired electron of Q(A)(-.) with two nitrogen nuclei in the protein matrix. From an analysis of the experimental data the nitrogen nuclear quadrupole resonance parameters were determined: e(2)qQ/h = 1.52 MHz, eta = 0.82 and e(2)qQ/h = 3.04 MHz, eta = 0.66, which are assigned to the N-14(8(1))-H group of His M219 and the peptide N-14 of Ala M260, The ESEEM spectrum of Q(A)(-.) in reaction centers of the Rb, sphaeroides mutant W(M252)Y shows that the nitrogen of Trp M252 is not interacting with Q(A)(-.), that of the mutant H(M266)C shows that manipulating the Fe binding site strongly affects the Q(A)-binding site. Show less
Roobol-Boza, M.; Shochat, S.; Tjus, S.E.; Hagman, A.; Gast, P.; Andersson, B. 1995
The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins... Show moreThe biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states. Show less
Vos, R. van der; Franken, E.M.; Sexton, S.J.; Shochat, S.; Gast, P.; Hore, P.J.; Hoff, A.J. 1995
In this work, the magnetic field effects (MFE) on the triplet yield in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides 2.4.1 and its Tyr M210 --> Trp mutant are compared. The MFE is... Show moreIn this work, the magnetic field effects (MFE) on the triplet yield in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides 2.4.1 and its Tyr M210 --> Trp mutant are compared. The MFE is measured between 25 and 225 K by monitoring the absorbance. Using monochromatic polarised and unpolarised light, linear dichroic (LD-)MFE curves were obtained. Simulations of the (LD-) MFE measured at low temperatures (25 K) are presented, which suggest that the exchange interaction in the mutant and in the wild type have opposite sign. This is explained by considering the free energy differences between the excited primary donor state P-1* and the charge-separated state. The magnetic field dependence of the MFE of Q-depleted mutant RCs at low magnetic fields is similar for temperatures between 25 and 225 K, implying that k(T) is practically temperature independent. This suggests that the temperature dependence of the triplet yield is due to a change in radical pair singlet recombination rate k(s). The exchange interaction between the oxidised primary donor and reduced intermediary bacteriopheophytin acceptor of mutant RCs is twice as large as that of the wild type. This is attributed to an upward change of the energy level of the charge-separated state caused by the mutation. Show less
Shochat, S.; Gast, P.; Hoff, A.J.; Boender, G.J.; Leeuwen, S. van; Liemt, W.B.S. van; ... ; Groot, H.J.M. de 1995
