The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (g_z) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. the tetranuclear iron-sulfur clusters can have their g_z axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.     

Spectroscopic characterization of the number and type of iron-sulfur clusters in NADH:ubiquinone oxidoreductase

The number and type of iron-sulfur clusters present in the NADH dehydrogenase of the mammalian respiratory chain were studied by a combination of low temperature magnetic circular dichroism (MCD) and quantitative electron paramagnetic resonance spectroscopies. MCD was used with the high molecular weight, soluble enzyme, and EPR was used with both the purified enzyme and Complex I (NADH:ubiquinone oxidoreductase). The results of the EPR experiments of the two types of preparations agreed with each other, as well as with the data in the literature for various types of membrane-bound preparations. The two methods gave concordant results showing the presence of one binuclear and of three tetranuclear NADH-reducible iron-sulfur clusters. Earlier studies using the cluster extrusion technique indicated a higher ratio of binuclear to tetranuclear clusters which may be explained by cluster interconversion during the extrusion process.     

Reduction of the iron-sulfur clusters in mitochondrial NADH:ubiquinone oxidoreductase (complex I) by EuII-DTPA, a very low potential reductant

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain. It contains a flavin mononucleotide to oxidize NADH, and eight iron-sulfur clusters. Seven of them transfer electrons between the flavin and the quinone-binding site, and one is on the opposite side of the flavin. Although most information about their properties is from EPR, the spectra from only five clusters have been observed, and it is difficult to match them to the structurally defined clusters. Here, we analyze complex I from bovine mitochondria reacted with a very low potential reductant, to impose a potential approaching -1 V. We compare the spectra with those from higher potentials and from the 24 kDa subunit and flavoprotein subcomplex, and model the spectra by starting from those with fewer components and building the complexity gradually. Spectrum N1a, from the 24 kDa subunit [2Fe-2S] cluster, is not observed in bovine complex I at any potential. Spectrum N1b, from the 75 kDa subunit [2Fe-2S] cluster, exhibits a lower potential than the N3, N4 and N5 spectra of three [4Fe-4S] clusters. In the lowest potential spectra an N5-type spectrum is observed at unusually high temperature (indicating a significant change to the cluster, or that two clusters have very similar g values), the relaxation rate of N1b increases (indicating that a nearby cluster has become reduced) and a new feature with an apparent g value of 2.16 suggests an interaction between two reduced clusters. The consequences of these observations for electron transfer in complex I are discussed.     

On the stoichiometry of the iron-sulphur clusters in mitochondrial NADH: ubiquinone oxidoreductase.

The concentration of the iron-sulphur (Fe-S) cluster 1b, present in complex I or soluble high-molecular-mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation-broadened signals of the Fe-S clusters 2-4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady-state kinetic and inhibitor-titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152-159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer.     

The orientation of the magnetic axes of the membrane-bound iron-sulfur clusters of spinach chloroplasts

Spinach chloroplast membranes were oriented onto mylar sheets by partial dehydration, and the orientation of the magnetic axes of membrane-bound paramagnetic clusters determined by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the reduced Rieske iron-sulfur cluster signal is of orthorhombic symmetry oriented with th gy = 1.90 axis orthogonal to the membrane plane and with the gz = 2.03 axis in the membrane plane; the gx-axis is undetectable, presumably due to its broadness. If the Rieske center is a two-iron iron-sulfur cluster, we conclude that the iron-iron axis lies in the plane of the membrane. Illumination reduces the two bound chloroplast iron-sulfur proteins known as Clusters A and B. Center A is oriented such that gx = 1.86 and gy = 1.94 lie at an angle of about 40, and gz = 2.05 is at approximately 25, to the membrane plane. There are two possible orientations of Cluster B depending on the set of g-values assigned to this cluster. For one set of g-values, gz = 2.04 and gx = 1.89 are oriented in the plane of the membrane while gy = 1.92 is orthogonal to the plane. Alternatively, gz = 2.07 and gy = 1.94 are oriented approximately 50 and 40 to the membrane plane respectively, and gx = 1.80 is in the plane of the membrane. An additional light-induced signal at g = 2.15 oriented orthogonal to the plane is currently unexplained, as are other membrane perpendicular signals seen at g = 2.3 and g = 1.73 in dark-adapted samples.     

The role of heme and iron-sulfur clusters in mitochondrial biogenesis, maintenance, and decay with age.

Mitochondria decay with age from oxidative damage and loss of protective mechanisms. Resistance, repair, and replacement mechanisms are essential for mitochondrial preservation and maintenance. Iron plays an essential role in the maintenance of mitochondria, through its two major functional forms: heme and iron-sulfur clusters. Both iron-based cofactors are formed and utilized in the mitochondria and then distributed throughout the cell. This is an important function of mitochondria that is not directly related to the production of ATP. Heme and iron-sulfur clusters are important for the normal assembly and for the optimal activity of the electron transfer complexes. Loss of mitochondrial cytochrome c oxidase (complex IV), integrity of mtDNA, and function can result from abnormal homeostasis of iron. We review the physiological role of iron-sulfur clusters and heme in the integrity of the mitochondria and the generation of oxidants.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

The vectorial orientation of human monoamine oxidase in the mitochondrial outer membrane.

1. The localization of monoamine oxidase in the mitochondrial outer membrane was studied in preparations of human liver mitochondrial and brain-cortex non-synaptosomal and synaptosomal mitochondria. 2. Immunochemical accessibility in iso-osmotic and hypo-osmotic mitochondrial preparations was used to localize the enzyme. 3. It was shown that the immunochemically accessible tyramine-oxidizing activity was distributed approximately equally on both surfaces of the membrane in human liver and brain-cortex non-synaptosomal mitochondria. However, the immunochemically accessible beta-phenethylamine-oxidizing activity was situated predominantly on the outer surface, and the immunochemically accessible 5-hydroxytryptamine-oxidizing activity was situated predominantly on the inner surface of the mitochondrial outer membrane in liver and brain-cortex non-synaptosomal mitochondrial preparations. 4. Considerable variation in the distribution of the enzyme in preparations of synaptosomal mitochondria was seen. 5. The simplest model consistent with our observations is that, in liver and brain-cortex non-synaptosomal mitochondria, the tyramine-oxidizing activity is distributed on both sides of the mitochondrial outer membrane, the beta-phenethylamine-oxidizing activity is located on the outer surface of the outer membrane and the 5-hydroxytryptamine-oxidizing activity is located on the inner surface of the mitochondria outer membrane.     

EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase segment of the respiratory chain in Paracoccus denitrificans

The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy. Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4. In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV. The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b. The pH-dependent P. denitrificans [2Fe-2S] cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a. Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system. The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7. The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts. We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster. The P. denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311).     

The orientation of transhydrogenase in the inner mitochondrial membrane of rat liver

The orientation of the transmembranous enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the nonspecific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Proteinase K treatment of mitoplasts converted the 110,000 transhydrogenase monomer into a single immunoreactive species having Mr 75,000. This proteolytic product is stable to further incubation with the protease. Treatment of submitochondrial particles with proteinase K resulted in the disappearance of the 110,000 monomer and the transient formation of an intermediate product with Mr 52,000. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. This model indicates that transhydrogenase (Mr 110,000) contains a core of proteolytically inaccessible proteins within the membrane (Mr 23,000) bounded by extramembranous domains on the matrix (Mr 52,000) and cytoplasmic (Mr 35,000) face of the inner mitochondrial membrane.     

The spatial relationships and structure of the binuclear iron-sulfur clusters in succinate dehydrogenase.

Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.     

The Saccharomyces cerevisiae mitochondrial succinate:ubiquinone oxidoreductase.

The Saccharomyces cerevisiae succinate dehydrogenase (SDH) provides an excellent model system for studying the assembly, structure, and function of a mitochondrial succinate:quinone oxidoreductase. The powerful combination of genetic and biochemical approaches is better developed in yeast than in other eukaryotes. The yeast protein is strikingly similar to other family members in the structural and catalytic properties of its subunits. However, the membrane domain and particularly the role of the single heme in combination with two ubiquinone-binding sites need further investigation. The assembly of subunits and cofactors that occurs to produce new holoenzyme molecules is a complex process that relies on molecular chaperones. The yeast SDH provides the best opportunity for understanding the biogenesis of this family of iron-sulfur flavoproteins.     

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.     

Hematoporphyrin-promoted photoinactivation of mitochondrial ubiquinol-cytochrome c reductase: selective destruction of the histidine ligands of the iron-sulfur cluster and protective effect of ubiquinone.

Purified ubiquinol-cytochrome c reductase of beef heart mitochondria is very stable in aqueous solution; it suffers little damage upon illumination with visible light under aerobic or anaerobic conditions. However, it is rapidly inactivated when the photosensitizer hematoporphyrin is present during illumination. The hematoporphyrin-promoted photoactivation is dependent on sensitizer dose, illumination time, and oxygen. Singlet oxygen is shown to be the destructive agent in this system. The photoinactivation of ubiquinol-cytochrome c reductase is prevented by excess exogenous ubiquinone, regardless of its redox state. This protective effect is not due to protein-ubiquinone interactions but to the singlet oxygen scavenger property of ubiquinone. Ubiquinone also protects against hematoporphyrin-promoted photoinactivation of succinate-ubiquinone reductase and cytochrome c oxidase. The photoinactivation site in ubiquinol-cytochrome c reductase is the iron-sulfur cluster of Rieske's protein. Two histidine residues, presumably serving as two ligands for the iron-sulfur cluster of Rieske's protein, are destroyed. No polypeptide bond cleavage is detected. Photoinactivation has little effect on the spectral properties of cytochromes b and c1 but alters their reduction rates substantially. this photoinactivation also causes the formation of proton-leaking channels in the complex. When the photoinactivated reductase is co-inlaid with intact ubiquinol-cytochrome c reductase or cytochrome c oxidase in a phospholipid vesicle, no proton ejection can be detected during the oxidation of their corresponding substrates.     

Kinetics of assembly of complex III into the yeast mitochondrial membrane. Evidence for a precursor to the iron-sulfur protein

Complex III immunoprecipitated from yeast cells labeled in vivo with [35S]sulfate or [3H]leucine contained seven subunits with molecular weights ranging from 15,000 to 47,000 when analyzed by electrophoresis on polyacrylamide gels. The subunit composition of the immunoprecipitates was identical with that of the purified complex III isolated from bakers' yeast suggesting that the antiserum recognizes the holoenzyme assembled properly in the membrane (Sidhu, A., and Beattie, D.S. (1982) J. Biol. Chem. 257, 7879-7886). Kinetic studies using double-labeled yeast cells followed by immunoprecipitation of complex III indicated that the subunits of the complex are assembled into the holoenzyme at very different rates. Cytochromes b and c1 and the 15,000-dalton subunit were the first polypeptides to be assembled into the complex with a half-time of labeling of 2.0-2.4 min. Core protein I and the iron-sulfur protein were inserted more slowly into the complex with a half-time of labeling of 4.6 and 5.3 min, respectively. Calculations of precursor pool sizes of the subunits indicated that for both core protein I and the iron-sulfur protein, there are large pools of precursors. The iron-sulfur protein was synthesized in vivo as a larger precursor polypeptide of molecular mass 28,000 Da. The precursor was subsequently cleaved, in a process requiring an energized mitochondrial inner membrane, into an intermediate form 1,500 Da larger than the mature subunit. The conversion of the intermediate to the mature form occurred in the inner mitochondrial membrane.     

Activation of isolated NADH:ubiquinone reductase I (complex I) from Escherichia coli by detergent and phospholipids. Recovery of ubiquinone reductase activity and changes in EPR signals of iron-sulfur clusters

NADH:ubiquinone oxidoreductase (NDH-1 or complex I) from Escherichia coli was purified using a combination of anion exchange chromatography and centrifugation in sucrose density gradient. The dependence of enzyme activity on detergent and phospholipids was studied. Artificial hexaammineruthenium reductase activity was not affected by dodecyl maltoside (DDM) and asolectin. Ubiquinone reductase activity had a bell-shape dependence on DDM concentration; 7-10-fold activation could be achieved. Treatment with asolectin subsequently yields additional 2-fold activation with a corresponding increase in the apparent V(max) and without significant changes in apparent K(m). Comparative EPR studies of complex I reduced with NADH, "as prepared" and "activated by asolectin" showed an increase in the signals derived mainly from two [4Fe-4S] clusters in the activated enzyme. One of these signals could be simulated with an axial spectrum with g values of g(xyz)= 1.895, 1.904, 2.05, which corresponds to the parameters reported for the N2 cluster. This data indicates conformational rearrangements of catalytic importance in complex I upon binding of phospholipids.