The coupling between electron transfer and Protein/Solvent Dynamics in Photosynthetic Reaction Centers: Spectroscopic Studies in Amorphous Matrices

We investigated at the molecular level protein/solvent interactions and their relevance in protein function through the use of amorphous matrices at room temperature. As a model protein, we used the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides, a pigment protein complex which catalyzes the light-induced charge separation initiating the conversion of solar into chemical energy. The thermal fluctuations of the RC and its dielectric conformational relaxation following photoexcitation have been probed by analyzing the recombination kinetics of the primary charge-separated (P+QA-) state, using time resolved optical and EPR spectroscopies. We have shown that the RC dynamics coupled to this electron transfer process can be progressively inhibited at room temperature by decreasing the water content of RC films or of RC-trehalose glassy matrices. Extensive dehydration of the amorphous matrices inhibits RC relaxation and interconversion among conformational substates to an extent comparable to that attained at cryogenic temperatures in water-glycerol samples. An isopiestic method has been developed to finely tune the hydration level of the system. We have combined FTIR spectral analysis of the combination and association bands of residual water with differential light-minus-dark FTIR and high-field EPR spectroscopy to gain information on thermodynamics of water sorption, and on structure/dynamics of the residual water molecules, of protein residues and of RC cofactors. The following main conclusions were reached: (i) the RC dynamics is slaved to that of the hydration shell; (ii) in dehydrated trehalose glasses inhibition of protein dynamics is most likely mediated by residual water molecules simultaneously bound to protein residues and sugar molecules at the protein-matrix interface; (iii) the local environment of cofactors is not involved in the conformational dynamics which stabilizes the P+QA-; (iv) this conformational relaxation appears to be rather delocalized over several aminoacidic residues as well as water molecules weakly hydrogen-bonded to the RC.

Coupling instrumentation and methodology in the search for traces of life on the early Earth and Mars

Evaluating the nature of the earliest, often controversial, traces of life in the geological record (dating to the Palaeoarchaean, up to ~3.5 billion years before the present) is of fundamental relevance for placing constraints on the potential that life emerged on Mars at approximately the same time (the Noachian period). In their earliest histories, the two planets shared many palaeoenvironmental similarities, before the surface of Mars rapidly became inhospitable to life as we know it. Multi-scalar, multi-modal analyses of fossiliferous rocks from the Barberton greenstone belt of South Africa and the East Pilbara terrane of Western Australia are a window onto primitive prokaryotic ecoystems. Complementary petrographic, morphological, (bio)geochemical and nanostructural analyses of chert horizons and the carbonaceous material within using a wide range of techniques – including optical microscopy, SEM-EDS, Raman spectroscopy, PIXE, µCT, laser ablation ICP-MS, high-resolution TEM-based analytical techniques and secondary ion mass spectrometry – can characterise, at scales from macroscopic to nanoscopic, the fossilised biomes of the earliest Earth. These approaches enable the definition of the palaeoenvironments, and potentially metabolic networks, preserved in ancient rocks. Modifying these protocols is necessary for Martian exploration using rovers, since the range and power of space instrumentation is significantly reduced relative to terrestrial laboratories. Understanding the crucial observations possible using highly complementary rover-based payloads is therefore critical in scientific protocols aiming to detect traces of life on Mars.