Giorgio Gennari

Synthesis of chlorophyll and photosynthetic competence in etiolated and greening seedlings of Larix decidua as compared with Picea abies

Summary The development of the photosynthetic apparatus was studied in seedlings of Picea abies and Larix decidua grown both in darkness and in light. In light the pattern of differentiation was similar in the two species, and mature chloroplasts showed a well developed thylakoid system arranged into granal and intergranal regions. In the dark, etio-chloroplasts were formed that had different lamellar and tubuliform membrane amounts in the two species. Spruce chloroplasts contained small prolamellar bodies and many thylakoids; in L. decidua chloroplasts, voluminous prolamellar bodies and only a few thylakoids were formed. Fluorescence emission spectra of cotyledons at 77 K as well as pigment analyses showed different behaviour of L. decidua compared with P. abies when grown in the dark. When etiolated seedlings were exposed to the light, the rate of phototransformation of the etio-chloroplast and the achievement of photosynthetic functionality was fast in spruce and slow in larch. Spruce cotyledons attained normal values of in vivo chlorophyll fluorescence characteristics, i.e. Fv/Fm and quenching coefficients qP and qNP within 1.5 hours of light exposure; with larch, the control value of qP was reached much more quickly (ca 3 h) than that of Fv/Fm (ca 20 h), indicating that PS I becomes fully active before PS II. After exposure to light, etiolated larch seedlings behave like those gymnosperms lacking the ability to synthesize chlorophyll in the dark.

FURTHER STUDIES ON THE CRYSTAL-VIOLET-SENSITIZED PHOTOOXIDATION OF CYSTEINE TO CYSTEIC ACID

Abstract—Crystal violet sensitizes the selective photooxidation of cysteine to cysteic acid; hydrogen peroxide is also formed as an end product. The participation of singlet oxygen in the photoreaction has been ruled out, since exposure of cysteine to this reagent, generated by chemical or photochemical processes, gives only cystine as a product. The photoreaction is inhibited by radical scavengers such as hydroquinone and allylic alcohol. A mechanism is proposed involving hydrogen abstraction by the triplet dye from the thiol group of cysteine.

Solvent effects on the emissive properties of trans-3-styrylquinoline in neutral and acidic solutions

Abstract The wavelength dependence of the fluorescence quantum yields of trans-3-styrylquinoline and of its protonated form has been studied in solvents of different polarity and hydrogen-bonding characteristics. The results obtained show that in all the examined solvents trans-3-styrylquinoline, in both neutral and protonated forms, exists as a mixture of two conformational isomers. The fluorescence lifetimes, emission and trans → cis photoisomerization quantum yields strongly depend on the solvent but they show no unequivocal correlation with its properties.

3-styrylquinoline conformers: A photophysical and photochemical study

Abstract The emission spectrum of a 10−6 M cyclohexane solution of trans-3-styrylquinoline varies with the excitation wavelength. Fluorescence excitation spectra at room temperature and at 77 K indicate the presence of two emitting species which are assumed to be conformational isomers arising from rotation about the single bond linking the styryl part of the molecule to the quinoline ring. The dependence of the photophysical and photochemical properties of trans-3-styrylquinoline in various solvents on the excitation wavelength are reported. The influence of pH on the composition of the conformers and the dependence on the excitation wavelength of the percentage of molecules which attain the prototropic equilibrium in the excited state were studied.

Paramagnetic metal ions as protectors of selected regions of protein molecules from photodynamic action.

Abstract The paramagnetic ion Co2+ and the diamagnetic ion Zn2+ were coordinated with the glutamyl-35 and aspartyl-52 residue at the active site of lysozyme. Upon irradiation with visible light in the presence of proflavin, Zn2+-lysozyme underwent total loss of enzymic activity as well as of the tryptophyl and methionyl side chains; the kinetics of photoinactivation and of tryptophan photooxidation were coincident with those determined for native lysozyme. On the contrary, irradiation of Co2+ lysozyme caused the specific modification of the peripheral tryptophan-63 and tryptophan-123, and a decrease of the enzymic activity to 84%. The photooxidized sample showed no appreciable distortion in the spatial conformation with respect to the unirradiated protein, thus explaining the retainment of a high catalytic efficiency. The strong inhibitory power of paramagnetic ions against the photodynamic action of dyes on proteins was also evidenced by our studies with ribonuclease A, whose complexes with Zn2+ or Cu2+ were subjected to proflavin-sensitized photooxidation over the pH range 5.8–8.2. At all pH values Zn2+-ribonuclease A displayed a marked photolability, whereas the presence of Cu2+ minimized or completely prevented both the impairment of the biological activity and the damage of the amino acid side chains. In particular, after irradiation at pH 6.9, we isolated a 72% active sample which was modified at methionine-79 and histidine-105. Conformational studies on the photooxidized derivatives of lysozyme and ribonuclease A, and the analysis of our results on the basis of the X-ray models of the native proteins demonstrated that the inhibitory action of Co2+ and Cu2+ occurs throughout almost the entire protein molecule, the efficiency of the protection being related to the distance of a given amino acid from the paramagnetic ion and the symmetry of the magnetic field. Besides providing a valuable tool for protecting enzymes from photodynamic inactivation, the aforesaid technique opens new prospects in the field of dye-sensitized photooxidation of proteins.

Probing the topography of proteins in solution by photosensitized oxidation. The heme environment in horse heart ferrocytochrome c.

Abstract Irradiation of ferrocytochrome c by visible light at pH 8.2 induced the photooxidation of Trp-59, as well as that of Met-80 and His-18, which are the two protein ligands for Fe 2+ . When the irradiation was performed at pH 11.1, Tyr-48 was also photooxidized. A comparison of the present data with those obtained upon irradiation of ferricytochrome c suggests that in the ferrous protein, the side chains of Tyr-48 and Trp-59 are closer to the heme system than in the ferric derivative.

Effect of the nature of aryl and heteroaryl groups on the excited state properties of asymmetric 1,4-diarylbutadienes

Abstract The excited state properties of a series of asymmetric 1-Ar,4-phenylbutadienes (Ar=naphthyl, phenanthryl, anthryl, pyrenyl, pyridyl and thienyl) have been investigated by stationary and pulsed techniques with the main aim of assessing the effect of the nature and size of the Ar group on the photophysical and photochemical behaviour. The comparison of the present results with those of previous studies on the corresponding 1,2-diarylethenes shows how the extension of the unsaturated chain affects the excited state properties and opens alternative adiabatic and “one photon–two bonds” isomerization mechanisms. The role of the relative position of the L a and L b states of Ar with respect to the olefinic B u state on the photobehaviour is discussed.

Conformational equilibrium in trans-aza-aryl-ethylenes: n-styrylquinolines and n-styrylisoquinolines

The ground state conformational equilibrium of trans-n-styrylquinolines (n=2, 3, 4, 6, 7 and 8) and n-styrylisoquinolines (n = 1 and 3) has been studied in polar and apolar solvents. The fluorescence and trans→cis photoisomerization properties show that the presence of the hetero- cyclic nitrogen does not modify the steric requirements for the stability of the possible conformers. The influence of the styryl position with respect to the heteroatom, and of the solvent on the equilibrium composition and on the photophysical behaviour of the individual rotamers has been particularly investigated.

PHOTOOXIDATION OF HORSE AND SPERM‐WHALE MYOGLOBIN SENSITIZED BY THE HEME GROUP

Abstract—The irradiation of horse and sperm‐whale Fe 3 * or Fe 2* myoglobins with visible light showed that axial ligands that render the heme diamagnetic (e.g. O2, CO or CN‐) endow the hemoproteins with a marked photosensitivity. In contrast, high‐spin myoglobins are unaffected by visible light. These findings appear to be of general validity for all hemo‐proteins and are in agreement with the involvment of the triplet state of the heme as the reactive intermediate. In all cases, the overall photoprocess occurs within a very narrow spatial range, leading to specific modification of these photooxidizable side chains adjacent to the chromophore. Therefore, this technique can be used to probe the environment of the prosthetic group in hemoproteins. In particular, our data suggest that, in horse myoglobin, histidines‐93 and ‐64 represent the heme‐linked and the distal imidazole groups, respectively; moreover, the thioether function of methionine‐131 must be nearer the heme in horse than in sperm‐whale myoglobin. The selectivity of the photoreaction can be further enhanced by a suitable choice of the sixth ligand, and/or by controlling the pH of the irradiated solution. For example, for both proteins, irradiation of the cyanide derivative results in specific photooxidation of the proximal histidine, whereas irradiation of horse CO‐ferromyoglobin at pH values below 6 causes specific photooxidation of methionine‐131. Consequently, this photooxidative procedure can also be utilized to monitor conformational changes upon binding of the heme with different ligands, as well as to achieve the selective modification of amino acid residues, which are usually buried inside the protein molecule.

Probing the topography of proteins in solution by photosensitized oxidation. The catalytic region of papain.

Abstract The thiol function of the single cysteinyl residue at the active site of papain was selectively conjugated with either the dinitrophenyl or the fluorescein thiocarbamyl group. Absorption, fluorescence and circular dichroism studies showed that, in both cases, the introduction of the chromophoric moiety caused no significant alterations of the spatial geometry characteristic of native papain. Irradiation of the dinitrophenyl derivative by visible light resulted in the specific photo-oxidation of histidine-159 and trytophan-177, which appeared to be the only potentially photo-oxidizable amino acids adjacent to the labelling group; their distance from the thiol function of cysteine-25 was evaluated to be about 5 A. These two residues do not appear to make an essential contribution to the structural stability of papain, since the oxidative modification of their side chains induced only limited modifications of the over-all conformation of the protein. On irradiation of the fluorescein-papain complex, the preferential photooxidation of tryptophan-177, histidine-159 and tryptophan-26 took place; in parallel, there was a drastic collapse of the tertiary structure of the protein molecule. It is concluded that tryptophan-26 is oriented in a direction different from that of tryptophan-177 and histidine-159; hence, this residue is probably not directly involved in the catalytic function of the enzyme. However, the intactness of its side chain is critical for maintaining the native three-dimensional structure of papain.