Piericiden A Sensitivity, Site 1 Phosphorylation, and Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase during Iron-limited Growth of Candida utilis

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir t

Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase, Piericidin Sensitivity, and Site 1 Phosphorylation in Different Growth Phases of Candida utilis

Reports in the literature indicate that during the exponential phase of growth of Candida utilis NADH oxidation is insensitive to rotenone, that rotenone sensitivity is acquired during the transition to the late stationary phase and is again lost on catabolite repression. The acquisition and loss of rotenone sensitivity appears to be accompanied by similar changes in Site 1 phosphorylation but does not appear to be reflected in the rate of oxidation of NADH (by mitochondria) or of NAD-linked substrates (by mitochondria or whole cells). In the present paper evidence is presented that these fluctuations in sensitivity to inhibitors of NADH oxidation reflect the presence of different types of inner membrane-bound NADH dehydrogenases in different phases of growth. Thus inner membrane preparations from exponential phase cells contain an NADH dehydrogenase which reacts equally well with ferricyanide and juglone as electron acceptor, appears to be very labile, and lacks EPR signals corresponding to iron-sulfur Centers 1 and 2, whereas a new species, probably an iron-sulfur protein, with resonances at g|| = 2.01, and g⊥ = 1.92 in the reduced state, is present. This species is not significantly reduced by NADH. In corresponding preparations from late stationary phase cells NADH-ferricyanide activity is high, juglone reductase activity is low, and the enzyme is stable and exhibits the EPR signals of iron-sulfur Centers 1 and 2, whereas the EPR signals of iron-sulfur Centers 3 + 4 change very little on transition from exponential to stationary phase cells. There is also a decrease in cytochrome concentration. Most prominent among these is a b-type cytochrome (g = 2.54; 2.23; 1.87) which decreases 2- to 3-fold. The EPR detectable species with g|| = 2.01 and g⊥ = 1.92 in the reduced state is no longer detected. On catabolite repression of late stationary phase cells there is an 80 to 90% decline in NADH-ferricyanide activity, of iron-sulfur Centers 1 and 2, a 50 to 60% decrease of Centers 3 + 4, and an increase in a b cytochrome, but the specific activity in NADH-juglone reductase and NADH oxidase assays increases, the enzyme becomes once again labile, and the EPR detectable species with g|| = 2.01 and g⊥ = 1.92 appears on reduction with dithionite. All these changes are prevented by cycloheximide. The data suggest that sensitivity to piericidin A and coupling to energy conservation Site 1 are properties of the type of NADH dehydrogenase present in late stationary phase cells but not in exponential phase or of catabolite-repressed cells

Di- and Trinuclear Mixed-Valence Copper Amidinate Complexes from Reduction of Iodine

Molecular examples of mixed-valence copper complexes through chemical oxidation are rare but invoked in the mechanism of substrate activation, especially oxygen, in copper-containing enzymes. To examine the cooperative chemistry between two metals in close proximity to each other we began studying the reactivity of a dinuclear Cu(I) amidinate complex. The reaction of [(2,6-Me2C6H3N)2C(H)]2Cu2, 1, with I2 in tetrahydrofuran (THF), CH3CN, and toluene affords three new mixed-valence copper complexes [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I3)(THF)2, 2, [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I) (NCMe)2, 3, and [(2,6-Me2C6H3N)2C(H)]3Cu3(μ3-I)2, 4, respectively. The first two compounds were characterized by UV-vis and electron paramagnetic resonance spectroscopies, and their molecular structure was determined by X-ray crystallography. Both di- and trinuclear mixed-valence intermediates were characterized for the reaction of compound 1 to compound 4, and the molecular structure of 4 was determined by X-ray crystallography. The electronic structure of each of these complexes was also investigated using density functional theory

Studies on mitochondria and submitochondrial particles by paramagnetic resonance (EPR) spectroscopy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32412/1/0000489.pd

Multiple frequency EPR studies on three forms of oxidized cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase (cytochrome aa3) (EC 1.9.3.1) has been demonstrated to occur in several forms when the redox centers in the protein are thought to be fully oxidized. We report here the results of extensive EPR studies at 3, 8.9, 9.2, 9.4, 15 and 34 GHz on the resting state, the alternative resting state (with g = 12 at 9 GHz) and pulsed state (with g = 5 signal at 9 GHz). Theoretical consideration is given to all binary spin-coupling possibilities under the constraint that the iron atoms are either ferric or ferrous and the copper atoms are either cupric or cuprous. We conclude that the g = 12 signal can arise from any spin system with S > 1 and |D| = 0.15 cm-1. The g = 5 signals originate from an excited, integer-spin system with |D| = 0.035 cm-1, which is approximately 7 cm-1 above the ground state (not observed in EPR). It is pointed out that in interpretations of data and elaboration of suitable models in this field, the implications of spin-coupling should be considered in a comprehensive and not in a selective way. At 3 GHz, EPR spectra of CuA in the resting, pulsed and anaerobically oxidized states show that this center is identical in its EPR for all three states.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25085/1/0000516.pd

Studies on the spin-spin interaction between flavin and iron-sulfur cluster in an iron-sulfur flavoprotein

When the di- or trimethylamine dehydrogenases (trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7) of certain methylotrophic bacteria are reduced by two electrons with substrate unusual EPR signals arise at g = 2 and g = 4 (Steenkamp, D.J. and Beinert, H. (1982) Biochem, J. 207, 233-239; 241-252) indicative of spin-spin interaction between the EMN and iron-sulfur compounds of these enzymes. An attempt is made to understand, describe and simulate these spectra in terms of a triplet state with possible contributions from both dipolar and anisotropic exchange (J) interactions. No direct measurement of J is available, but various approaches to setting limits to J are outlined. According to these, J [approximate] 0.4 to 3 cm-1 or 15 to 50 cm-1. The spectra show, in the g = 2 region, a pair of rather sharp inner and a pair of broad outer lines; the latter broaden as well as move out from the center with increasing time (after substrate addition) and substrate concentration, while there is little change of g = 4. The best fits to such a spectra were obtained by assuming distribution of D and E values, depending on substrate effects and arriving presumably from `g-strain'. The fact that both shapes and intensities at g = 2 and g = 4 could be reproduced simultaneously at two frequencies indicates that the assumptions underlying our approaches and interpretations are permissible and reasonable, although we cannot claim their uniqueness. The distance between the centers of the spin densities of the flavin radical and the Fe-S cluster is thought to lie between the limits 3 to 5 A if the asymmetries in the spin-spin interaction are magnetic dipole-dipole in origin. Because there is an indication that the interaction is anisotropic exchange, the upper limit is less stringent.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26294/1/0000379.pd

Comments on "the interpretation of the EPR and Mossbauer spectra of two-iron, one-electron iron-sulfur proteins"

Evidence is presented indicating that the note mentioned in the title is incorrect in two important aspects. 1.) According to our results of ENDOR spectroscopy and computer simulations of Mossbauer spectra, the conclusions drawn are based on an erroneous interpretation of the Mossbauer spectra. 2.) According to quotations from the literature, previous interpretations of experimental data on iron-sulfur proteins are incorrectly represented.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33508/1/0000005.pd

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs