Giovanni Venturoli: The career of a real scholar

The first time I met Giovanni was in 1975 when he decided to follow my Biophysics lectures as a student of the Degree in Physics. He immediately demonstrated a very sharp mind and a keen interest in science. I therefore was quite pleased when he expressed his intention to work in our lab for the final experimental thesis required to complete his degree. He graduated in 1977 and presented a thesis on electric phenomena occurring in energy transducing membranes of photosynthetic bacteria, at that time a main interest in our research group. His degree work, innovative and clear-cut, was published immediately as a research paper, the first of a long series to follow (Melandri et al., 1978).

After graduation he continued to work in our laboratory and his contributions proved very soon to be essential. He improved very effectively our fast-spectrophotometry apparatus, and very patiently set up experimental conditions for very accurate measurements of light induced ATP synthesis in bacterial chromatophores during short flashes of light (Melandri et al., 1980). He showed a very marked skill in experimental work together with an excellent ability for an accurate analysis of the experimental data. In the following I will review briefly his impressive research achievements from the early 1980s to 2025, grouping them according to the topic addressed. Due to the complexity of the scientific contents, this summary is grossly oversimplified and the pertinent literature by other laboratories is omitted. The reader should refer to the papers cited in this review, restricted to the most important contributions by Venturoli, and to the vast reference lists herein.

In the following years other research fields were investigated: the effect of UQ 10 extraction or enrichment, varying thereby the size of UQ 10 endogenous pool, on the electron transport from the photosynthetic reaction center to the cytochrome bc 1 complex (Venturoli et al., 1986); the kinetics of charge recombination from the reduced primary (Q A.-) or secondary (Q B.-) electron accepters to the oxidized bacteriochlorophyll reaction center (P +) and the effects of the antennae complex on its rate (Dezi et al., 2007); the evaluation of the intrinsic buffer capacity of bacterial chromatophores, a crucial aspect for quantitative studies of ATP synthesis (Turina et al., 1990).

Another small, but very significant group of papers concerned the reconstitution of the entire cyclic electron transport chain, starting from purified reaction center (bRC) and cytochrome bc 1 complexes of Rhodobacter capspulatus, incorporated in liposome enriched in UQ 10 and internally containing cytochrome c 2. These works were the results of the collaboration between our and the laboratory headed by Prof. Dieter Oesterhelt, Max-Planck-Institute of Biochemistry, Martinsried (Germany) in which Nadia Gabellini, a former member of our group, was working (Venturoli et al., 1990a; Venturoli et al., 1990b). This research was eventually completed by the demonstration that the reconstituted electron transport system was competent in promoting the synthesis of ATP, when purified ATP synthetase was inserted in the liposome bilayer with the proper orientation (Gabellini et al., 1989). Many other experimental works, related to the photosynthetic electron transport chains in bacteria other than Rb. capsulatus, such as Chromatium vinosum, Rhodoferax fermentans and Rb. veldkampii, were the results of collaborations with different laboratories.

The role of UQ 10 in bacterial photosynthesis has been of constant interest in Giovanni’s research. An important example is the study of several bc 1 mutant in which Lys329 in the cytochrome b subunit was replaced by different amino-acids; this study demonstrated that a positively charged residue was required for an efficient activity of the Q o site of the complex, the site for the oxidation of reduced UQ 10 (UQ 10 H 2) and the consequent release of 2 H + (Francia et al., 2019). This work was the result of a collaboration with the laboratory of Fevzi Daldal, Dept. of Biology, Univ. of Pennsylvania, Philadelphia (USA), who produced systematically such mutants. These were further utilized rather extensively in Bologna for research utilizing different experimental approaches (see below).

A relevant group of papers applied X ray absorption spectroscopy (XFAS, EXFAS) to the study of metal binding sites in photosynthetic bacterial complexes. These works were carried out in collaboration with Federico Boscherini, a colleague in the Physics Department in Bologna and utilized the European Synchrotron Radiation Facility in Grenoble. Their experimental approach is well described in a convincing methodological paper (Giachini et al., 2010). An example of these works is the comparative study of the inhibitory Zn 2+ -binding sites in bacterial, avian end bovine bc 1 complexes (Giachini et al., 2007), suggesting in different structures (particularly unlike in bacterial and bovine bc 1) a comparable mechanism of inhibition due to the binding of Zn 2+ to an His residue and competing with its role as a H + donor/accepter. Such mechanism was also proposed for the analogous inhibition by Zn 2+ of the H + entry into the Q B site of the photosynthetic reaction center (Giachini et al., 2005). A third example is the study of the coordination shell of the native non-heme Fe 2+ atom present between the Q A and Q B (bound to one Glu and four His residues) and the effect of dried trehalose matrices on the bond lengths (Veronesi et al., 2008). A common theme in these works is a more detailed knowledge of the pathways for the protonic traffic from a proton-releasing (or to a proton-accepting) site to (or from) the external medium. Using the same experimental approach other metal binding sites in different enzymatic complexes were studied: in bacterial transhydrogenase of Escherichia coli (in collaboration with the laboratory of J.B. Jackson University of Birmingham, UK), and in bovine cyt c oxidase (in collaboration with the laboratory of S. Papa, University of Bari Italy).

The clarification of the functional role and of the structural features of the Puf X protein is another very noteworthy group of publications. puf X is a gene included int the puf operon of Rb. sphaeroides encoding for the two subunits (L and M) of the bRC and the two subunits (α and β) of the inner antenna complex LH1, plus a gene involved in bacteriochlorophyll synthesis. The role of the Puf X protein was obscure at the time of this research. The first hints on its role were described in two early papers confirming that this protein was necessary for photosynthetic anaerobic growth (Barz et al., 1995a) and for an efficient UQ 10 /UQ 10 H 2 exchange under multiple turnover conditions (Barz et al., 1995b) (again in collaboration with the laboratory of D: Oesterhelt, in which Francesco Francia, at that time working in Martinsried, had a major role). A decisive turning point was reached when it was discovered that the purified bRC-LH1 complex could be fractionated into two different sedimentation bands on a sucrose gradient,. The two bands contained respectively a monomeric and a dimeric form of the complex and Puf X was required for the formation of the dimer (Francia et al., 1999). It was therefore proposed that the presence of the dimer was essential for an efficient exchange of UQ 10 /UQ 10 H 2 between the Q B site of the bRC and the soluble UQ 10 pool. The final demonstration of this proposal was obtained when the low-resolution structure of the dimer was published, obtained by cryo-electron-microscopy of bidimensional crystals (Scheuring et al., 2004). This structure revealed a S-shaped dimer containing two core complexes formed by one bRC and a 12 LH 1 heterodimers and joined by a central Puf X dimer. Each core complex had an opening allowing the fast exchange of UQ 10 /UQ 10 H 2 from and to the Q B site of the bRC. This structure was perfectly consistent with the functional features both in vivo and in isolated membranes. The function of Puf X was thus established as an inhibitor of the complete closing of the LH1 ring around the bRC complex and as the fusion agent for the dimerization, thereby allowing the UQ 10 traffic from and to the Q B site of the bRC.

After this rather short survey of Giovanni’s research over more than 20 years the last group of papers here discussed is concerned with the dynamics of proteins embedded in trehalose glasses. This topic has been by far the most important in the last part of his career and produced no less than 25 papers. This research was prompted by the peculiar properties of a desert plant (resurrection plant, Craterostigma plantagineum) capable of withstanding extreme dehydration (an other extreme stress conditions such as very high or very low temperatures) even over months or years, and restarting its vegetative cycle after watering. This type of latent life, common in other plants and animals is defined as anhydrobiosis. This plant was shown to synthesize high concentrations of the disaccharide trehalose (α-D-glucopyranosyl-α-D-glucopyranoside), a feature common to many other organisms resistant to drought. The premises of this research were lawn with the kinetic analysis of the non-exponential rate of re-reduction of the oxidized photosynthetic reaction center (P +) from the primary (Q A −.) or secondary (Q B −.) quinone accepters after a flash of light (a charge recombination reaction between a radical pair), studied as a function of temperature in UQ 10 -containing lipid vesicles (Palazzo et al., 2000). The trans-membrane electron transport chain in Rb. capsulatus (or Rb sphaeroides) RC is formed by a dimer of bacteriocholophylls (P), placed near the extracellular side of the membrane and two branches, bound to the two main protein subunits (L and M), each formed by a bacheriochorophyll, a bacteriopheophitin and converging to Q A. These cofactors are positioned in such a way that the positive P +. and the Q A −. radicals are placed on opposite membrane sides. Q A −. reduces a second UQ 10 (Q B), promoting its complete reduction to UQ 10 H 2,after two turnovers. The reduction of UQ 10 or the oxidation of UQ 10 H 2 involves the uptake or the release of 2 H +, a crucial event for energy conservation and ATP synthesis. The radical P +. can be reduced either by Q A −. or Q B −. in charge recombination reactions. The multi-exponential kinetics of these reactions has been interpreted as evidence for the existence of many different pathways related to motions of the protein matrix (Palazzo et al., 2000).

Based on the results in (Palazzo et al., 2000) a large amount of the following research on the behavior of proteins embedded in trehalose glasses utilized the P +. Q A −. (or P +. Q B −.) recombination reaction in purified photosynthetic reaction centers as a model for probing the effects of trehalose (and of other sugars) on protein dynamics. With time a very solid model for the protein-trehalose interactions was proposed. The mains steps of this extensive research are here briefly described.

The first two important works on this topic (Palazzo et al., 2002; Francia et al., 2003) demonstrated that inclusion of bRC in trehalose matrices had very marked effects of the kinetics of the P + Q A − and the P + Q B − charge-recombination reactions. These papers were the first of a long-standing collaboration and a sincere reciprocal esteem with Prof. Lorenzo Cordone, Univ. of Palermo, Italy, whose studies on the effect of trehalose matrices on myoglobin, suggested the extension of such research to a photosynthetic system. In these works it was shown that the progressive desiccation of trehalose matrices, up to 97% trehalose/H 2 O, markedly accelerates the rate of charge recombination from Q A − (Palazzo et al., 2002) and blocks the electron transfer between the primary and the secondary quinones (Francia et al., 2003). It was concluded that the electron transfer from Q A − to Q B is rate-controlled by the mobility of the protein (coupled to the protonation of Q B − and in line with the displacement of Q B, induced by light, from a distal to a proximal site with the respect to the dark adapted structure, documented by X-ray diffraction studies). The stabilization of P + Q A −, on the other hand, was proposed to be related to a different mechanism (Francia et al., 2003). The acceleration of the charge recombination of P + Q A − was observed only with trehalose and was matched by a protective effect of this sugar against thermal degradation. It was proposed that the protein dynamics is slowed and eventually blocked by the interaction with the external sugar/H 2 O matrix, most likely through a H-bond network involving interfacial water. This effect occurs with trehalose but not with sucrose, since in sucrose/H 2 O matrices a “nanophase separation” takes place, causing a less efficient protective effect (Palazzo et al., 2008).

This research was also complemented by a study on the dependence of the charge recombination rate of P + Q A − or of P + Q B − on the activity of water, modulated by changing the concentrations of different co-solutes. The results indicated that the activity of water affects the P + Q A − Q B –P + Q A Q B − equilibrium implying that this equilibrium is coupled the release of protein-bound water (Francia et al., 2008).

Q A −. and Q B −., being free radicals, could be studied by EPR spectroscopy. This aspect of research was approached in a very fruitful collaboration with Prof. Klaus Möbius, Fachbereich Physik, Freie Universität Berlin, Berlin, and Prof. Wolfgang Lubitz, Max-Planck-Institut für Chemische Energiekonversion, Mülheim, Germany, with whom Giovanni established a very solid friendship. In these studies the matrix humidity was controlled by means of an equilibrium with specific saline solutions, thereby attaining an important improvement for the accuracy of the experimental conditions (Malferrari et al., 2014). The phenomena in trehalose matrices, as well as in other sugar/H 2 O solutions, were studied directly on endogenous Q A −. and Q B −. radicals and on the P +. Q B −. radical pair (Savitsky et al., 2010), or using a nitroxide spin probe, dissolved in the matrix (Malferrari et al., 2014) or attached covalently to protein residues in the H subunit of the bRC (Nalepa et al., 2017), a subunit exposed to the aqueous solvent. The results confirmed, on the whole, the hypothesis described above, since the vitreous matrix did not perturb the distance or the orientation of the internal native radicals (Savitsky et al., 2010), but clearly affected the properties of spin labels dissolved in the matrix or placed at the protein/solvent interface (Nalepa et al., 2017).

These researches were extended to the photosynthetic reaction centers of oxygenic photosynthesis, namely, of a cyanobacterial photosystem I (PSI) (Malferrari et al., 2016), and spinach photosystem II (PSII), the latter completed or deprived of the water oxidizing complex (WOC) (Mamedov et al., 2021). These works were performed in collaboration with the laboratory of a long-standing friend, Alexey Semenov, Moscow State University, Moscow, Russia (Malferrari et al., 2016; Mamedov et al., 2021) as well as with Lubitz’s laboratory (Malferrari et al., 2016).

The photosystems in oxygenic photosynthesis differ substantially from the bacterial anoxygenic ones:

PSII is resembling bRC in electron accepter site, since it contains two quinone (Q A and Q B, in this case two plastoquinones) that are reduced by the electron released by a chlorophyll a dimer (P 680), through two parallel chains of intermediates (chlorophylls a and pheophytins) converging on Q A. The electrons reducing P 680 +. are originated from the oxidation of water, catalyzed by a prosthetic group (the water oxidation complex,WOC), consisting of a Mn 4 CaO 5 cluster shielded by membrane extrinsic subunits (Malferrari et al., 2016). The transfer of electrons from the WOC to P 680 +. is mediated by a redox-active tyrosine (Y z, i.e., Y 161 in the D1 subunit); the ensuing positive radical Y z +. radical appears to be stabilized by an interacting His-190.

PSI, on the contrary, resembles bRC in electron donor side: the pathway of the electron transport starts from a chlorophyll a dimer (P 700) and follows two parallel mediator chains (each containing a chlorophyll a (A 0) and a quinone (A 1 a phylloquinone)) that converge into a 4F4S center (F x). F x reduces then a pair of 4F4S centers (F A /F B) bound the a membrane extrinsic subunit. F A /F B interacts with soluble ferredoxin at the stromal side. The physiological reductant of P 700 +. is plastocyanine, a soluble copper protein in the thylakoid lumen (or in other cases cytochrome c 2, in photosunthetic bacteria, or cytochrome c 6, in Cyanobacteria).

In both PSII and PSI a transmembrane radical pair is formed upon photoexcitation with various electron accepter (A 1, F x or F A /F B in PSI; Q A or Q B in PSII). In the absence of external electron accepters and donors the positive holes in P 680 +. or in P 700 +. can be reduced in charge-recombination reactions, taking place with different reaction rates and sensitivity to dehydration. In PSI electrons can be transferred from A 1 -. (τ = 0.1 ms), from F x −. (τ = 1–10 ms) and from F A /F B −. (τ = 0.06–0.5 s). In PSII charge recombination from Q A −. occurs with τ = 0.17–1.2 ms; P 680 +. can be reduced also by Y z. in a bi-exponential reaction. (τ = 4–20 μs in WOC depleted PSII).

In both photosystems the results demonstrated that upon dehydration the multi-exponential kinetics of charge recombination was profoundly modified: in PSI the fast phases (from A 1 - or F x −.) included more than 85% of the reaction rates in dry matrices, while in hydrated matrices or in solution the slow rates (from F A /F B −.) prevailed. In PSII preparation, deprived of the WOC complex and of the Q B plastoquinone, the reduction of P 680 +. by Y z was delayed in dry matrices and increased to 50% of the total. The contribution of the recombination reaction Q A −. to P 680 +. to the overall rate of reduction, almost negligible in solution and in well hydrated matrices, gradually increased to the remaining 50%. As a whole these results were interpreted as evidence that the interaction with a sugar-water matrix hindering the protein motions to which the intraprotein redox reactions are coupled: in PSII the reverse reaction F A /F B −. to F x and in PSII the reduction of P 680 +. by Y Z and the P 680 +. Q A −. charge recombination. Likewise in PSII the electron transport between Q B −. and Q A are blocked (Kaminskaya et al., 2003). These redox events (except the P 680 +. Q A −. reaction) occur in rather peripheral regions of the protein where the interaction with the ambient should be stronger. The kinetic phases mainly slowed by the matrix interaction appear often to involve protonation/deprotonation events. Another interesting observation in Malferrari et al. (2016) was that the water content in glasses containing PSII samples was systematically much higher than that containing bRC or PSI with the same degree of humidity, suggesting that the amount of interfacial water is larger in the former case; this is in agreement with the structure of PSII that includes a greater number of membrane extrinsic subunits, while the intramembrane subunits are shielded by a detergent ring.

Finally Venturoli’s laboratory contributed substantially to a very extensive study on the protein CAHS D of tardigrade Hypsibius exemplaris (Mamedov et al., 2021). Tardigrades are microscopic animals (more than 1000 species) surviving desiccation and other environmental stresses (included the exposure outside the Space Lab!), entering a state of suspended life (anhydrobiosis) in a way analogous to other organisms accumulating trehalose or other sugars. The transition into anhydrobiosis required the expression of specific proteins CAHS, of which CAHS D is a typical specimen. This protein can form a gel protective to the cellular structures. The functional properties of this protein has been studied using a variety of approaches the required the cooperation of 12 different scientific institutions. Bologna contributed with the functional characterization of the system again utilizing the charge recombination P +. Q A −. bRC as an experimental tool. The presence CAHS D in bRC samples desiccated at controlled humidity values produced the same effects previously observed in trehalose containing glasses, i.e., the acceleration of the P +. Q A −. charge-recombination rate. Thus, the formation of gels appears to generate a protective shell at the protein-matrix interface as proposed for the trehalose matrix. Other experimental evidence reported in (Sanchez-Martinez et al., 2024), including mutagenesis, scanning microscopy, Small Angle X-ray Scattering, protein structure prediction, in vitro and in vivo experiments and many other approaches, brought to the conclusion that the formation of a CAHS D gel inside the cell was strictly related to, if not the main cause, for the survival in dry conditions. The structural model proposed and demonstrated experimentally was the formation of self-assembling filaments, similar to the intermediate filaments of the cytoskeleton, and formed by CAHS proteins and induced by dehydration. The gel condensation inside the cell upon desiccation was correlated to a decreased metabolic rate. Both events were reversed upon rehydration.

On the basis of these extensive researches a model was proposed in which trehalose forms in dry glasses an amorphous matrix at very low H 2 O/sugar ratios (Mamedov et al., 2021). This matrix interacts with hydrophilic portions of proteins, and other biological macromolecules, slowing down their dynamics, very often essential for their functional events. In extreme dryness a complete block of mobility can be reached. These phenomena also protect cellular structures from thermal degradation, or other types of damage, since the immobilization of the biological structures prevents the loss of the native folding and activity. The sugar molecules do not form direct bonds (water replacement model), but the interaction is mediated by the hydration shell, present in the fully hydrated state, and persisting at the molecule/matrix interface (water entrapment and anchorage model) (Mamedov et al., 2021). Upon re-hydration the matrix dissolves, a mobile hydration shell is re-formed and full functionality is re-established.

The essence of anhydrobiosis is therefore closely related to the formation of an internal matrix of trehalose (or other disaccharides) or by suitable proteins (CAHS) that immobilize and protect the cellular structures under conditions of drought. This protection holds also for other types of stress. These phenomena appear to take place in parallel with a drastic decrease of the metabolic rate, most likely due to the block of the dynamics of enzymes.

This is a concise review of the impressive scientific achievements of Giovanni Venturoli. Even at a superficial reading his papers stem up for their high quality: meticulous descriptions of the experimental methods, precise report of the results obtained, very sophisticated treatments of the experimental data and very deep and thorough discussions and conclusions. It is also evident his open-mindedness for the use of a vast range of experimental techniques and for the search of fruitful collaborations with other laboratories. These outstanding qualities go together with a very kind disposition toward collaborators and students and a very thoughtful attitude.

As for most young researches, the first steps of his academic career have been slow and laborious. After a fellowship in Bologna he had to travel, for 2 years, as far as Calabria, in the tip of Italy, to teach “Fudamental Physics” in the University of Cosenza. He returned to Bologna in 1984 and began his regular career as Technician (1984). In 1991 he was nominated Associate Professor of Photobiology in the Faculty of Science (Biology) and moved to the position of Associate Professor of Applied Physics in 1994. He has taught “Fundamental Physics” (Degree in Biology); “Spectroscopic Methods in Biology” (Master of Biology) and “Photobiology” (Master in Photochemistry). During his long scientific activity he has exerted an intensive and much appreciated seminar activity, both in Italy and abroad. He has always been coherent with his view of science, up to the point of refusing advances in his academic career since they did not appeared to him in line with his biophysical background. Unfortunately in the academic world not always the scientific integrity is an advantage!

On a more personal note, other qualities should be mentioned: Giovanni is an inveterate reader; he plays piano and is a fine connoisseur of music, his favorite composer being J.S. Bach; he is also an expert in art, having for all his life practiced oil painting and etching; he is also interested in paleontology, since when, as a young boy, he was wandering in the hills of Bologna searching for fossils or prehistoric artifacts. He also loves nature, mountains and, last but not least, dogs and cats. As I wrote in the title of this review he is therefore an all-round scholar, rarely met our modern society. I was lucky to have him as a student, a collaborator and, mainly, as a friend.