Structure of the P700(+ )A1(-) radical pair intermediate in photosystem I by high time resolution multifrequency electron paramagnetic resonance: analysis of quantum beat oscillations.

The geometry of the secondary radical pair P700(+)A1(-), in photosystem I (PSI) from the deuterated and 15N-substituted cyanobacterium Synechococcus lividus, has been determined by high time resolution electron paramagnetic resonance (EPR), performed at three different microwave frequencies. Structural information is extracted from light-induced quantum beats observed in the transverse magnetization of P700(+)A1(-) at early times after laser excitation. A computer analysis of the two-dimensional Q-band experiment provides the orientation of the various magnetic tensors of with respect to a magnetic reference frame. The orientation of the cofactors of the primary donor in the g-tensor system of is then evaluated by analyzing time-dependent X-band EPR spectra, extracted from a two-dimensional data set. Finally, the cofactor arrangement of P700(+)A1(-) in the photosynthetic membrane is deduced from angular-dependent W-band spectra, observed for a magnetically aligned sample. Thus, the orientation of the g-tensor of P700(+) with respect to a chlorophyll based reference system could be determined. The angle between the g1(z) axis and the chlorophyll plane normal is found to be 29 +/- 7 degrees, while the g1(y) axis lies in the chlorophyll plane. In addition, a complete structural model for the reduced quinone acceptor, A1(-), is evaluated. In this model, the quinone plane of is found to be inclined by 68 +/- 7 degrees relative to the membrane plane, while the P700(+)-A1(-) axis makes an angle of 35 +/- 6 degrees with the membrane normal. All of these values refer to the charge separated state, observed at low temperatures, where forward electron transfer to the iron-sulfur centers is partially blocked. Preliminary room temperature studies of P700(+)A1(-), employing X-band quantum beat oscillations, indicate a different orientation of A1(-) in its binding pocket. A comparison with crystallographic data provides information on the electron-transfer pathway in PSI. It appears that quantum beats represent excellent structural probes for the short-lived intermediates in the primary energy conversion steps of photosynthesis.

[The vital staining of nuclear chromatin and the chromosomes of HeLa cells with the fluorochrome AMHA binding to DNA]. / Uber die Vitalfluorochromierung des Kernchromatins und der Chromosomen von HeLa-Zellen mit AMHA und die Bindung des Acridinfarbstoffs an DNA.

The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations CF less than or equal to 10(-4) M and short incubation periods tI less than or equal to 2 h cell growth is not affected by the drug. But at higher CF and longer tI the population doubling time of the cell cultures rapidly increases, and finally the cells die. In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm-1 between the 0-0-transitions of the long wave length 1La absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm-1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission. Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nucleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant KF1 = 1.1.10(5) M-1, binding parameter n1 = 0.15; the pre-intercalative or external binding 2, KF2 = 6.9.10(5) M-1, n2 = 0.21; the external binding 3, KF3 = 2.8.10(5) M-1, n3 = 0.55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant KF1 = 1.1.10(5) M-1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, KF1 = (1-4).10(4) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)