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ABSTRACTThe mitochondrial matrix of the yeast Saccharomyces cerevisiaecontains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in ?ssq1mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389–18393, 1998). However, the intermediate form in both wild-type and ?ssq1mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in ?ssq1mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of ?ssq1cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in ?ssq1mitochondria. ?ssq1mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.

The mitochondrial matrix of the yeast Saccharomyces cerevisiaecontains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Δssq1mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389–18393, 1998). However, the intermediate form in both wild-type and Δssq1mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Δssq1mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Δssq1cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Δssq1mitochondria. Δssq1mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.

To understand the role of the [4Fe-4S]2+cluster in controlling the activity of the Escherichia colitranscription factor FNR (fumarate nitrate reduction) during changes in O2availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S]2+cluster of FNR-L28H in the presence of O2because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S]2+to [2Fe-2S]2+cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S]2+cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S]2+cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S]2+cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S]2+cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.

FNR is a global regulator that controls transcription of genes whose functions facilitate adaptation to growth under O2limiting conditions. It has long been appreciated that the activityof FNR must be regulated by O2availability, since FNR dependent gene expression is observed in vivo only under anaerobic conditions, while similar levels of this protein are present in both aerobic and anaerobic grown cells. Recent progress in this field has shown that anaerobically purified FNR contains a [4Fe‐4S]2+cluster and that this [4Fe‐4S]2+cluster is sufficiently unstable toward O2to make it suitable as an O2sensor. The presence of the [4Fe‐4S] cluster increases dimerization of FNR which is correlated with an increase in site‐specific DNA binding of FNR, a property expected of transcription factors of the FNR/CRP family. According to Mössbauer spectroscopy on purified FNR and cells containing overexpressed FNR, the [4Fe‐4S]2+cluster of FNR is converted by O2to a [2Fe‐2S]2+in high yield. The [2Fe‐2S]2+cluster can be reconverted to the [4Fe‐4S]2+cluster on reduction with dithionite in vitro raising the possibility that the [2Fe‐2S]2+cluster is a biologically inactive intermediate which may be more readily available for reconstitution into the [4Fe‐4S]2+form than the Fe‐free apoform. The ability to observe, by Mössbauer spectroscopy, the Fe‐S clusters of FNR in cells containing high levels of FNR should be of value in further unraveling how FNR functions in vivo. Attempts to reduce the [4Fe‐4S]2+cluster of FNR with dithionite indicated that the redox potential of the +1/+2 couple is ≤−650 mV and that the [4Fe‐4S]+cluster form is, therefore, not likely to occur in vivo.

In the past five to ten years, it has become increasingly apparent that the function of Fe-S clusters is not limited to electron transfer, a function implicit in their discovery. We now know that the vulnerability of these structures to oxidative destruction is used by nature in sensing O2, iron, and possibly also nitric oxide. Changes in the oxidation state of Fe-S clusters can also serve as a reversible switch.

Significant experimental observations and concepts leading from various sources toward today's bioenergetics are briefly sketched. To limit the essay to manageable proportions main consideration is given to origins in research on metabolism and biological oxidations and attendant energy conversions. Relevant data and dates are summarized in table form.

In this brief survey, the path of development of our knowledge of the iron‐sulfur enzyme aconitase [citrate(isocitrate)hydrolyase EC4.2.1.3.] is traced from its discovery in 1937, Particular emphasis is on developments in the past decade, when EPR, Mössbauer and electron nuclear double resonance spectroscopies, X‐ray crystallography, and mutational analysis were applied to the problem. More recently discovered was the significant amino acid sequence identity between mitochondrial aconitase and the iron regulatory factor or iron‐responsive element binding protein (IRE‐BP). This has led to the realization that IRE‐BP is an alternative form of cytosolic (not of mitochondrial) aconitase that is devoid of its cubane Fe‐S cluster.— Beinert, H., Kennedy, M. C. Aconitase, a two‐faced protein: enzyme and iron regulatory factor. FASEB J.7: 1442‐1449; 1993.

This review considers recent information, in the following order, on complex proteins containing Fe‐S clusters together with other prosthetic groups, on hydrogenases, unexpected functions of Fe‐S clusters, novel cluster types, protein structures furnishing the cluster ligands, evolutionary aspects, and spectroscopic methods and theoretical approaches used or developed in the study of Fe‐S proteins.—Beinert, H. Recent developments in the field of iron‐sulfur proteins. FASEB J.4: 2483‐2491; 1990.

Crystal structures of mitochondrial aconitase with the inhibitors trans-aconitate and nitrocitrate bound to the [4Fe-4S] cluster have been solved and refined at 2·05 Å resolution with R-factors of 0·168 and 0·172, respectively. Crystallization of aconitase with the substrates citrate and cis-aconitate has not been possible because the enzyme turns over and selects enzyme with isocitrate bound into the crystal lattice. Therefore we have analyzed crystal structures of the enzyme complexed with inhibitor analogs of these two substrates. The structure with nitrocitrate bound provides a model for citrate binding. The structure with trans-aconitate bound provides a model for cis-aconitate binding in two ways: Fe4 of the [4Fe-4S] cluster is five-coordinate and the carbon at the Cβposition is trigonal. These results allow the model for the reaction mechanism to be extended to all three natural substrates of aconitase. The results support a model in which citrate and isocitrate form similar chelate structures related by 180° rotation about the Cα-Cβbond while the intermediate cis-aconitate binds in either of two ways (citrate mode or isocitrate mode). In both inhibitor complexes a H2O molecule is also bound to Fe4. In the structure with nitrocitrate bound, partial occupancy of sulfate in the active site is observed accompanied by hydroxyl binding to Fe4. Comparison of the structures with isocitrate, trans-aconitate, nitrocitrate and sulfate bound reveals preferred orientations for the three types of oxygens ligated to Fe4 (carboxyl, hydroxyl and H2O) supporting the proposed roles for His101, Asp165 and His167 in the catalytic mechanism.

A comparison of the effects of adrenocorticotropic hormone (ACTH) on the cholesterol side chain cleavage system of zona glomerulosa and zona fasciculata-reticularis tissue of the rat adrenal is presented. Following hypophysectomy, the rate of pregnenolone formation from endogenous cholesterol in mitochondria from zona fasciculata-reticularis was lower than in mitochondria from zona glomerulosa. Also, the proportion of high spin, cholesterol-bound side chain cleavage cytochrome P-450 was smaller, as measured by spectrophotometry and electron paramagnetic resonance spectroscopy.

The criteria of homogeneity or native state of a protein are prone to become ambiguous when applied to membrane proteins, such as cytochrome‐coxidase, which are purified by extraction with detergents. Properties of the purified material depend on the detergent used and on details of the purification protocol followed with any single batch of a preparation. We present arguments to show that the evidence presently available in published form does not justify the designation [(1987) J. Biol. Chem. 262 3160–3164] of one type of preparation as being closer to the native state than others.

The criteria of homogeneity or native state of a protein are prone to become ambiguous when applied to membrane proteins, such as cytochrome- coxidase, which are purified by extraction with detergents. Properties of the purified material depend on the detergent used and on details of the purification protocol followed with any single batch of a preparation. We present arguments to show that the evidence presently available in published form does not justify the designation [(1987) J. Biol. Chem. 262 3160–3164] of one type of preparation as being closer to the native state than others.

Digitonin - fractionated photosystem - I subchloroplasts were titrated potentiometrically between -450 and -610 mV at pH 10. Examination of the titrated subchloroplasts by low-temperature (13°K) electron paramagnetic resonance spectroscopy revealed resonances centered at values of 2.05, 1.94, 1.92, 1.89, and 1.86 on the g-factor scale. The peak heights depended on the potentials at which the chloroplasts were poised. The resonances of at least three iron-sulfur centers can be recognized: one with lines at g= 2.05 and 1.94; one with lines at g= 2.05, 1.92, and 1.89; and one for which only a line at g= 1.86 has been resolved. The midpoint potentials of the iron-sulfur species fall into two distinctly separate regions: the titration profile of the g= 1.94 signal, the first segment of the g= 2.05 plot, and the rise phase of the g= 1.86 signal had a value of -530 ± 5 mV; the upper segment of the g= 2.05 plot, the decrease phase of the g= 1.86 signal, and the g= 1.89 profile had a midpoint potential estimated to be [unk] -580 mV. The oxidation-reduction reaction of each of the bound iron-sulfur species, as represented by the changes of the electron paramagnetic resonance spectra, was reversible and apparently involved a two-electron change.Titration at pH 9 could only be carried to -560 mV, and essentially only the first half of the titration behavior as found at pH 10 was seen. At any given potential more positive than -560 mV, the part of the iron-sulfur protein that was not reduced electrochemically could be reduced photochemically, but only to the maximum extent reduced electrochemically at -560 mV. Whereas, chloroplasts illuminated at room temperature and then frozen while still being illuminated developed a signal similar to that produced by electrochemical reduction at -610 mV, illumination at 77°K did not bring about photoreduction beyond that accomplished electrochemically at about -560 mV.Dithionite alone in the dark and under anaerobic conditions brought about a partial reduction to the extent of the first electrochemical reduction step. Dithionite plus illumination at room temperature or dithionite plus methyl viologen in the dark produced the maximum signal. Electron paramagnetic resonance spectra due to either light or electrochemically reduced iron-sulfur proteins showed no detectable decay for at least 3 days when samples were stored in the dark at 77°K.

The electron acceptors in ubihydroquinone-cytochrome creductase (Complex III) derived from beef heart mitochondria were investigated by low temperature electron paramagnetic resonance spectroscopy at ≤20 K and optical reflectance spectroscopy at 100 K. Three resonances were resolved in the region where the low field lines of low spin ferric heme compounds are usually found. These lines were assigned to cytochromes c1(gz= 3.33), bk(gz= 3.44), and bt(gz= 3.78) on the basis of correlation with optical spectra during reduction. A broad line typical of the center line (gy) of a low spin ferric heme is seen at g ∼ 2. Other EPR-detectable electron acceptors in Complex III are the iron-sulfur center previously detected by EPR at 77° (J. S. Rieske, D. H. MacLennan, andR. Coleman(1964) Biochem. Biophys. Res. Commun.15, 338); a species whose signal appears at g = 1.93 on reduction, presumably an iron-sulfur protein, and possibly due to contamination with succinate-ubiquinone reductase (Complex II); two acceptors of unknown structure and function, one with a signal centered at g = 2.01 (oxidized form) and the other with lines at g = 2.04 and 1.99. The signal of the latter is absent in the oxidized state, appears on partial reduction and disappears on complete reduction, indicating that three discrete oxidation states may be involved.

The EPR-detectable (≥4.2 K) electron acceptors of the DPNH-ubiquinone reductase segment of the electron transfer system of beef heart mitochondria were studied in preparations of varying complexity. Four distinct iron-sulfur centers associated with this segment of the electron transfer system were identified. They occur in approximately equal concentrations, and this concentration approximates that of the bound flavin of DPNH dehydrogenase. Quantitative reductive titrations with DPNH and dithionite showed that the order of the oxidation-reduction potentials of the ironsulfur centers is: Center 3 ≥ 2 >> 4 > 1, indicating that there are two centers of high and two of low potential. Complex I accepts approximately 20 electron neq per mg of protein from DPNH. In rapid reaction studies using the freeze-quench method the rates of reduction of the iron-sulfur centers by DPNH could not be resolved. With acetylpyridine DPNH, however, the rates of appearance of the signals were in this sequence: Center 2 > 3 + 4 > 1.

The copper x-ray fluorescence excitation spectrum of cytochrome coxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) has been recorded in the 245—270 K range. The beat pattern observed in the extended x-ray absorption fine structure can be accounted for only by postulating a combination of sulfur and nitrogen (or oxygen) ligands to the copper. The average Cu—S distance is 2.27 ± 0.02 Å and the average Cu—N (or Cu—O) distance is 1.97 ± 0.02 Å. The amplitudes require ca, 1-1.5 sulfurs and 2 nitrogens (or oxygens) per copper. The distribution of sulfur ligands between CuAand CuBsites is not known, although there is some evidence that two sulfur atoms are bound to CuA.

The transcription factor FNR from Escherichia coliregulates transcription of genes in response to oxygen deprivation. To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo. This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen. Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster. To determine the type of Fe-S cluster present, an oxygen-stable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein. Based on the sulfide analysis, the stoichiometry (3.3 mol of S2−/FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clusters per mutant FNR monomer. However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one [4Fe-4S] cluster present per monomer of FNR LH28-DA154. We assume that the wild type also contains one [4Fe-4S] cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy. Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile. In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer. The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding. Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.

Reports in the literature indicate that during the exponential phase of growth of Candida utilisNADH 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 bcytochrome, 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.

Ethanolamine ammonia-lyase catalyzed the adenosylcobalamin-dependent deamination of 2-aminopropanol as well as that of ethanolamine. When the enzyme-coenzyme complex was frozen in liquid nitrogen while it was acting on 2-aminopropanol, a large, well defined EPR spectrum was demonstrated. The spectrum consisted of two components: a broad signal at g = 2.34, and an asymmetrical doublet with g values of 2.04 and 1.99. The over-all signal corresponded to about 1.3 spins per active site, the electron density being approximately equally divided between the species producing the g = 2.34 component and the one represented by the asymmetrical doublet. The configuration of the signal was the same regardless of whether the incubation was conducted aerobically or anaerobically. Both components of the signal were saturated by increasing power, the doublet being the more easily saturated component. EPR spectroscopy at two different microwave frequencies (X band and K band) showed that the doublet represented a single paramagnetic species whose signal was split by an interaction with another paramagnetic species in its vicinity.

Beef heart aconitase contains 3Fe clusters in its inactive and 4Fe clusters in its active form. The fully active form can be restored from the inactive one by insertion of Fe2+, whereas S2-is not required. Chemical analyses for iron and labile sulfide yield Fe/S2-ratios of 0.66-0.74 for the inactive and 0.90-1.03 for the active form. Sulfane sulfur (S0) was not detected. We propose on the basis of these data that the inactive form may arise from the active one by loss of one iron only per cluster with the sulfur remaining as S2-in a [3Fe-4S] structure. Measurements by extended x-ray absorption fine structure (EXAFS) spectroscopy on the 3Fe form of aconitase yield a Fe··S distance of 2.24 Å and a Fe··Fe distance of 2.71 Å. This Fe··Fe distance is in agreement with that obtained by EXAFS on ferredoxin II of Desulfovibrio gigas, another 3Fe protein, but disagrees with Fe··Fe distances observed for the 3Fe cluster of Azotobacter vinelandiiferredoxin I by x-ray diffraction—namely, 4.1 Å. We suggest that this difference may be due to the presence of a [3Fe-3S] structure in the Azotobacterferredoxin I crystals vs. a [3Fe-4S] structure in liquid or frozen solutions of aconitase. The [3Fe-3S] cluster has been shown to have a relatively flat twist-boat structure, whereas a [3Fe-4S] cluster could be expected to essentially maintain the compact structure of the [4Fe-4S] cluster. This would explain the differences in Fe··Fe distances. Two possible structural models for a [3Fe-4S] cluster are discussed.

Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitrostudy of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g= 2.02 signals of the [3Fe-4S] clusters and the g≈ 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical,g∥= 2.11 and g⊥= 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g≈ 2.04 species and in the case of mitochondrial aconitase, a transient signal atg≈ 2.032 appeared. This signal is tentatively assigned to the d9form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g= 2.02 [3Fe-4S] proteins.

The locus of inhibition of nicotinamide adenine dinucleotide, reduced form (NADH) oxidation in mitochondria by rotenone, piercidin A, and barbiturates is considered in the light of available information. Most lines of evidence indicate that the point of inhibition is on the O2side of NADH dehydrogenase. Kinetic experiments on the substrate-induced appearance of the electron paramagnetic resonance signal at g= 1.94 in membrane preparations (ETP) reveal that these inhibitors do not interfere with the reduction of the electron paramagnetic resonance detectable iron by NADH. Our spectrophotometric studies on complex I give no evidence for absorbance differences between untreated and rotenone or piericidin inhibited preparations, which can be attributed to nonheme iron. Whatever changes were observed appear to be due to cytochromes. These experiments, therefore, do not support the idea that in inhibited preparations electron transport is interrupted between the flavin and nonheme iron components of NADH dehydrogenase.The specific binding of rotenone and piericidin seems to involve both lipid and protein. The possibility that NADH dehydrogenase participates in the binding is suggested by the apparent stoichiometric relation between specific binding site titer and NADH dehydrogenase content and the profound effect of mersalyl inhibition of the enzyme on piericidin binding capacity.ETP, electron transport particle.

During the anaerobic reductive titration of both C. pasteurianumferrodoxin and milk xanthine oxidase, two different EPR signals ascribable to iron were seen to arise in succession. More than one species of iron is therefore involved in electron transfer in each one of these proteins.

On the basis of oxidoreductive rapid kinetic and titration experiments with purified cytochrome coxidase (EC 1.9.3.1), monitored by electron paramagnetic resonance (EPR) at 13°K and by spectrophotometry at 100°K, a new assignment of EPR signals is proposed. The bulk of both the low-spin (g= 3.0; 2.2; 1.5) and highspin (g= 6; 2) signals is attributed to the component with the properties of traditional cytochrome a. It is further proposed that the absorption band at 655 nm represents the most unambiguous manifestation of the a3component.

Attention is drawn to a mechanism of redox control of gene expression involving Fe‐S proteins which depends on the disassembly and reassembly of Fe‐S clusters rather than a change in oxidation state. Iron Regulatory Protein (IRP)/aconitase and FNR are discussed as examples for such a mechanism.