Search

The interaction between gold sub-nanometer clusters composed of ten atoms (Au10) and tetrakis(4-sulfonatophenyl)porphyrin (TPPS) was investigated through various spectroscopic techniques. Under mild acidic conditions, the formation, in aqueous solutions, of nanohybrid assemblies of porphyrin J-aggregates and Au10 cluster nanoparticles was observed. This supramolecular system tends to spontaneously cover glass substrates with a co-deposit of gold nanoclusters and porphyrin nanoaggregates, which exhibit circular dichroism (CD) spectra reflecting the enantiomorphism of histidine used as capping and reducing agent. The morphology of nanohybrid assemblies onto a glass surface was revealed by atomic force microscopy (AFM), and showed the concomitant presence of gold nanoparticles with an average size of 130 nm and porphyrin J-aggregates with lengths spanning from 100 to 1000 nm. Surface-enhanced Raman scattering (SERS) was observed for the nanohybrid assemblies.

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.

Anions induce collapse of acid-denatured cytochrome c into a compact state, the A-state, showing molten globule character. Since structural information on partially folded forms of proteins is important for a deeper understanding of folding mechanisms and of the factors affecting protein stabilization, in this paper we have investigated in detail the effects of anions on the tertiary conformation of the A-state. We have found that the salt-induced collapse of acid-denatured cytochrome c leads to a number of equilibria between high-spin and low-spin heme states and between two types of low-spin states. The two latter states are characterized by conformations leading to a native-like Met-Fe-His axial coordination and a bis-His configuration. The equilibrium between these two A-states is dependent on the concentration and/or size of the anions (i.e. the bigger the anion, the greater its effect). Further, on the basis of fast kinetic data, a kinetic model of the folding process from the acid-unfolded protein to the A-state (at low and high anion concentration) is described. [ABSTRACT FROM AUTHOR]

The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O2 affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated in vitro by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O2 affinity (P50 = 2.7 torr) that was increased by S-nitrosation (P50 = 1.7 torr), whereas in tuna Mb, O2 affinity (P50 = 0.9 torr) was independent of S-nitrosation. O2 dissociation rates (koff) of trout and salmon Mbs were not altered when Cys were in the SNO or N-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O2 affinity by raising the O2 association rate (kon). Taken together, these results indicate that O2-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O2 entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb in vivo is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions.