Twenty years of biophysics of photosynthesis in Padova, Italy (1984-2005): a tale of two brothers.

This paper tells the history of two brothers, almost a generation apart in age, who met again, after having followed different academic paths, to introduce biophysical research in photosynthesis at the University of Padova. The development of two research groups, one in the Chemistry Department, the other in the Biology Department led to a comprehensive interdisciplinary group across academic barriers. The group of Giovanni Giacometti developed in Physical Chemistry, during the years before his retirement, with some roots which can be traced to the famous Linus Pauling school of the mid 1950s, and made possible, by the work of many students (especially Donatella Carbonera and Marilena Di Valentin) and of an older associate (Giancarlo Agostini). The group participated quite actively with a number of European and American laboratories in the application of physical techniques, especially Electron Spin Resonance (EPR) associated with Optical Spectroscopy (Optically Detected Magnetic Resonance; ODMR), and contributed to the development of the understanding of the structure-function relationships in photosynthetic membrane complexes, stimulated by the determination of the X-ray structure of the purple photosynthetic reaction center in the mid 1980s ( J. Deisenhofer, H. Michel, R. Huber and others). The younger brother of Giovanni, Giorgio Mario Giacometti, came to Padova after obtaining biochemical knowledge from the Rossi-Fanelli school in Rome, where Jeffries Wyman, Eraldo Antonini and Maurizio Brunori were the world masters of hemoglobin research. In Padova, together with a group of young scientists (at first Roberto Bassi and Roberto Barbato, now leaders of their own groups in Verona and in Alessandria respectively, followed soon by brilliant coworkers such as Fernanda Rigoni, Elisabetta Bergantino and more recently Ildikò Szabò and Paola Costantini), Giorgio approached more biochemical themes of oxygenic photosynthesis, such as purification and characterization of antenna chlorophyll-protein complexes, Photosystem II (PS II) particles and subunits, having always in mind structural and molecular problems at the level of the largest integrated particles, which are more difficult to investigate in detail by the spectroscopic techniques.

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers.

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).

Structural investigation of oxidized chlorosomes from green bacteria using multifrequency electron paramagnetic resonance up to 330 GHz.

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c (.+) [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316-325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a (.+) [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973-6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.

Optically detected magnetic resonance of intact membranes from Chloroflexus aurantiacus. Evidence for exciton interaction between the RC and the B808-866 complex.

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808-866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808-866 antenna complex. This evidence was provided by the comparison of the T-S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.