F. Bonomi

Bacterial frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS

Frataxin is an essential mitochondrial protein whose reduced expression causes Friedreichs ataxia (FRDA), a lethal neurodegenerative disease. It is believed that frataxin is an iron chaperone that participates in iron metabolism. We have tested this hypothesis using the bacterial frataxin ortholog, CyaY, and different biochemical and biophysical techniques. We observe that CyaY participates in iron-sulfur (Fe-S) cluster assembly as an iron-dependent inhibitor of cluster formation, through binding to the desulfurase IscS. The interaction with IscS involves the iron binding surface of CyaY, which is conserved throughout the frataxin family. We propose that frataxins are iron sensors that act as regulators of Fe-S cluster formation to fine-tune the quantity of Fe-S cluster formed to the concentration of the available acceptors. Our observations provide new perspectives for understanding FRDA and a mechanistic model that rationalizes the available knowledge on frataxin.

Head space sensor array for the detection of aflatoxin M1 in raw ewe's milk.

A novel screening method was developed for simple and rapid detection of aflatoxin M1 contamination in raw ewes milk samples without the need for sample pretreatment. The method was based on the use of a commercial head space sensor array system constituted by 12 metal oxide semiconductor sensors, 10 metal oxide semiconductor field-effect transistor sensors, and a pattern recognition software. Twenty-four raw milk samples collected from two different groups of ewes fed with a formulated feed that contained increasing amounts of aflatoxin B1 and six noncontaminated ewes milk samples were analyzed. The results obtained by using the head space sensor array, processed by statistical methods, made it possible to group the samples according to the presence or the absence of aflatoxin M1. Sample classification was in complete agreement with the aflatoxin M1 content measured by an enzyme-linked immunosorbent assay procedure. This is the first report, to our knowledge, of detection of aflatoxin M1 in ewes milk by a multisensor array.

Insertion of sulfide into ferredoxins catalyzed by rhodanese

In previous work [1] we studied the interaction of rhodanese (EC 2.8.1.1) an enzyme present in mitochondria, chloroplasts and bacteria, with the i ron sulfur flavoprotein succinate dehydrogenase (EC 1.3.99.1). Rhodanese catalyzes the transfer of sulfane sulfur from a sulfane containing anion to a thiophilic anion. Intermediate in this reaction is sulfur-substituted rhodanese ([S] rhodanese) which can be isolated. When some dithiol-containing compounds serve as acceptors, e.g., lipoate or dithiothreitol, the persulfide formed by rhodanese breaks down in an internal redox reaction yielding a disulfide and inorganic sulfide [2]. We found that the sulfane sulfur of [3SS]rhodanese or that of thiosulfate was transferred to succinate dehydrogenase which bound it as sulfide and had its iron-sulfur structure modified. In a comparative assay with other proteins the presence of the iron-sulfur structure appeared important for sulfide binding [ 1 ]. In the present work we have addressed ourselves to the problem of whether reduction and binding of sulfur was peculiar to succinate dehydrogenase or whether it also occurred with other iron-sulfur proteins. The reaction mechanism was also investigated. Since succinate dehydrogenase contains both a Fe4S4 structure of the Hipip type and two spinach ferredoxinlike Fe2S2 centers [3,4], we assayed spinach ferredoxin and ferredoxin from Clostridium pasteurianum which contains two Fe4S4 clusters per molecule.

Sulfide insertion into spinach ferredoxin by rhodanese

Abstract The behavior of non-aminoacidic protein-bound sulfur in the interaction between rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) and spinach ferredoxin was studied in presence or absence of thiosulfate, the substrate of rhodanese. The amount and oxidation state (sulfide, persulfide, trisulfide) of the sulfur on both proteins were determined. It appeared that sulfur is transferred from rhodanese to ferredoxin and also that the reverse transfer occurs. The amount transferred and the oxidation state of sulfur depend on the condition of interacting proteins and on the presence or not of thiosulfate, which drives rhodanese in its action as a transferase. Optical and circular dichroism spectra indicate an improved absorbance and ellipticity of the iron-sulfur centers of ferredoxin when this protein accepts sulfur from rhodanese. Rhodanese is in part modified and inactivated in the process. The decay is considerably diminished by thiosulfate. No transfer takes place to proteins which in their native state do not have iron-sulfur structure. The reactions outlined fit into the general scheme of the sulfur-transfer interactions of rhodanese with iron-sulfur proteins. Individual steps of transfer, however, specially as concerns reduction of persulfide sulfur of rhodanese to sulfide, appear to be specific for the acceptor protein considered.

A study of surface hydrophobicity of milk proteins during enzymic coagulation and curd hardening.

The formation of hydrophobic sites on the surface of casein micelles as a consequence of rennet action has been followed through the binding rate of a fluorescent probe and its distribution between a free and an ‘aggregated’ protein fraction. The variation of this parameter has been related to clotting time and curd hardening kinetics. Results show that a first aggregation of casein through hydrophobic sites interaction began as soon as rennet was added to milk. At the natural pH of milk, the sol-gel transition occurred when all the casein micelles were already involved in large aggregates. This was not the case with slightly acidified milk (pH 6·5 and 6·3) where clotting occurred well before the first aggregation step had been completed. The surface hydrophobicity of casein continued to increase in the curd due to continuing enzymic action and structural rearrangements. When this process has been completed the hardening of curd proceeds at an accelerated pace until it reached its maximum asymptotic value.

On the role of the 2Fe-2S cluster in the formation of the structure of spinach ferredoxin

Abstract The conformational changes of spinach ferredoxin in the apoprotein ⇌ holoprotein conversion have been studied by the far-ultraviolet circular dichroic technique. Apoferredoxin shows a largely disordered structure in solution. Evidence is presented, based on the effects either of chemical removal or of enzymic insertion of the 2Fe-2S center, that the cluster itself acts as the major force in determining the structure of spinach ferredoxin. The presence of sodium chloride cannot reverse the loss of structure consequent to the removal of the cluster, but stabilizes the residual structure once the cluster has been removed. Upon urea treatment of the apoferredoxin all the residual structural elements are lost even in the presence of sodium chloride.

Shelf life of case-ready beef steaks (Semitendinosus muscle) stored in oxygen-depleted master bag system with oxygen scavengers and CO2/N2 modified atmosphere packaging

This study aims to evaluate the stability of beef from Semitendinosus muscle packaged in oxygen permeable wrapped-tray units and stored in a master bag system, with and without oxygen scavengers. Changes in the atmosphere composition, microbiological indexes, myoglobin forms and color parameters were monitored during the storage in master bag, blooming and display life. The presence of scavengers reduced rapidly the oxygen concentration and maintained it at values not detectable instrumentally. Within few days of storage in master bags, the resolution of the transient discoloration was completed and the meat quality was maintained over the anoxic storage. After the removal from master bags meat bloomed completely reaching OxyMb level and Chroma values higher than those on fresh meat at t(0). During 48 h of display life at 4 °C, quality attributes had a decay slower than samples stored traditionally in air. Without scavengers the oxygen caused the irreversible discoloration within 7 days, due to the formation of metmyoglobin on the surface.

The inhibition of rhodanese by lipoate and iron-sulfur proteins

A study was made on the effects of DL-dihydrolipoate, lipoate and iron-sulfur proteins on the activity of rhodanese (EC 2.8.1.1) with dihydrolipoate or cyanide as acceptors. DL-Dihydrolipoate inactivates rhodanese, lipoate does not, and the opposite occurs with the sulfur-free form of the transferase. The observed effects vary with the sulfane sulfur acceptor from rhodanese (i.e., dihydrolipoate or cyanide) and depend on intramolecular oxidation of the catalytic sulfhydryl or on formation of a mixed disulfide with dihydrolipoate. Thiosulfate protects against inactivation by reloading the active-site cysteine with persulfide sulfur. The inhibition of sulfur transfer by iron-sulfur proteins appears related to the amount of native iron-sulfur structure interacting with rhodanese. The implications of the results for a possible biological role of rhodanese are considered.

The circular dichroism and optical absorbancy of the histidyl flavin during active—non-active transition of soluble succinate dehydrogenase

Gxaloacetate is a negative effector for succinate dehydrogenase having a major role in the process of activation, i.e., the reversible transition of the enzyme between a catalytically inactive and an active form. The stable form of the non-active enzyme is a complex with oxaloacetate, in a 1: 1 ratio to &win, and the unchanged modulator is dislodged only by denaturation of the protein or by any type of activation [l-4]. In [4] it was proposed that the substrate binding site and the regulatory site are not the same and oxaloacetate does not form a thiohemiacetal linkage with an SH group essential for enzymic activity as suggested [5,6] but it induces a change in conformation of the enzyme. The nature of the non-active form of the enzyme was deduced from reductive activation titrations [7]. Quantitative analyals of such titrations [

Sulfhydryl and disulfide content of succinate dehydrogenase

I led to the cormhrsion that the redox potential of the flavin in the active enzyme equala 6 * 20 mV (compatible to act as oxidant with respect to succirrate), On the other hand the redox potential of the flavin in none active enzyme is -186 f 20 mV. Such a low redox potential is compatible with that of free flati (-167 mV for the isolated histidyl flavin of succinate dehydrogenase [9]) and accounts for the fact that non-active enzyme can not be reduced by succinate unless the enzyme undergoes activation. The shift in the redox potential of the flavin was attributed to a ~o~o~a~n change of the flavin moiety 183, such aa bendiug of the planar form of oxidized fl O] or semiquinone [I I ] flavin to the nonplanar structure of reduced flavin [IO]. Molecular orbital calculationsindicated that the energy associated with such transformation would suffice to shift the redox potential by such an amount. A different mechanism accountig for such a shift, and which alao accounts for the fact that activation by reduction is a one electron reduction IS], is by interaction of polar groups with Nr or Ns of the isoalloxaxiue ring fl2]. Whatever is the mechanism shifting the potential of the flavin, both predict a major change in its CD spectrum. The isoalloxazme ring is devoid of optical activity unless asymmetry is introduced by interaction with the ribityl hydroxyls or with protein side chains [13], As documented here, a soluble, purified reconstitutively active succinate dehydrogenaae demon &rates major changes in its flavin (and FeS) CD spectra when transformed between its a&&e and nonactive states.