Andrej Musatov

Susceptibility of mitochondrial electron-transport complexes to oxidative damage. Focus on cytochrome c oxidase

Abstract Reactive oxygen species (ROS) are associated with a number of mitochondrial disorders. These include: ischemia/reperfusion injury, Parkinsons disease, Alzheimers disease, neurodegenerative diseases, and other age-related degenerative changes. ROS can be generated at numerous sites within the cell, but the mitochondrial electron transport chain is recognized as the major source of intracellular ROS. Two mitochondrial electron-transfer complexes are major sources of ROS: complex I and complex III. Oxidative damage to either of these complexes, or to electron transport complexes that are in close proximity to these ROS sources, e.g., cytochrome c oxidase, would be expected to inhibit electron transport. Such inhibition would lead to increased electron leakage and more ROS production, much like the well-known effect of adding electron transport inhibitors. Recent studies reveal that ROS and lipid peroxidation products are effective inhibitors of the electron-transport complexes. In some cases, inactivation of enzymes correlates with chemical modification of only a small number of unusually reactive amino acids. In this article, we review current knowledge of ROS-induced alterations within three complexes: (1) complex IV; (2) complex III; and (3) complex I. Our goal is to identify “hot spots” within each complex that are easily chemically modified and could be responsible for ROS-induced inhibition of the individual complexes. Special attention has been placed on ROS-induced damage to cardiolipin that is tightly bound to each of the inner membrane protein complexes. Peroxidation of the bound cardiolipin is thought to be particularly important since its close proximity and long residence time on the protein make it an especially effective reagent for subsequent ROS-induced damage to these proteins.

Glutathione peroxidase 4 differentially regulates the release of apoptogenic proteins from mitochondria

Glutathione peroxidase 4 (Gpx4) is a unique antioxidant enzyme that repairs oxidative damage to biomembranes. In this study, we examined the effects of Gpx4 on the release of various apoptogenic proteins from mitochondria using transgenic mice overexpressing Gpx4 [Tg(GPX4(+/0))] and mice deficient in Gpx4 (Gpx4+/- mice). Diquat exposure triggered apoptosis that occurred through an intrinsic pathway and resulted in the mitochondrial release of cytochrome c (Cyt c), Smac/DIABLO, and Omi/HtrA2 in the liver of wild-type (Wt) mice. Liver apoptosis and Cyt c release were suppressed in Tg(GPX4(+/0)) mice but exacerbated in Gpx4+/- mice; however, neither the Tg(GPX4(+/0)) nor the Gpx4+/- mice showed any alterations in the levels of Smac/DIABLO or Omi/HtrA2 released from mitochondria. Submitochondrial fractionation data showed that Smac/DIABLO and Omi/HtrA2 existed primarily in the intermembrane space and matrix, whereas Cyt c and Gpx4 were both associated with the inner membrane. In addition, diquat exposure induced cardiolipin peroxidation in the liver of Wt mice; the levels of cardiolipin peroxidation were reduced in Tg(GPX4(+/0)) mice but elevated in Gpx4+/- mice. These data suggest that Gpx4 differentially regulates apoptogenic protein release owing to its inner membrane location in mitochondria and its ability to repair cardiolipin peroxidation.

Ferricytochrome c protects mitochondrial cytochrome c oxidase against hydrogen peroxide-induced oxidative damage

An excess of ferricytochrome c protects purified mitochondrial cytochrome c oxidase and bound cardiolipin from hydrogen peroxide-induced oxidative modification. All of the peroxide-induced changes within cytochrome c oxidase, such as oxidation of Trp(19,IV) and Trp(48,VIIc), partial dissociation of subunits VIa and VIIa, and generation of cardiolipin hydroperoxide, no longer take place in the presence of ferricytochrome c. Furthermore, ferricytochrome c suppresses the yield of H(2)O(2)-induced free radical detectable by electron paramagnetic resonance spectroscopy within cytochrome c oxidase. These protective effects are based on two mechanisms. The first involves the peroxidase/catalase-like activity of ferricytochrome c, which results in the decomposition of H(2)O(2), with the apparent bimolecular rate constant of 5.1±1.0M(-1)s(-1). Although this value is lower than the rate constant of a specialized peroxidase, the activity is sufficient to eliminate H(2)O(2)-induced damage to cytochrome c oxidase in the presence of an excess of ferricytochrome c. The second mechanism involves ferricytochrome c-induced quenching of free radicals generated within cytochrome c oxidase. These results suggest that ferricytochrome c may have an important role in protection of cytochrome c oxidase and consequently the mitochondrion against oxidative damage.

Tryptophan 334 Oxidation in Bovine Cytochrome c Oxidase Subunit I Involves Free Radical Migration

A single tryptophan (W334(I)) within the mitochondrial‐encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W334(I) is converted to hydroxytryptophan as identified by reversed‐phase HPLC‐electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS–PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W334(I) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear‐encoded subunits, W48(IV) and W19(VIIc), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003–1009). Two aromatic‐rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface‐exposed tryptophans, where they produce hydroxytryptophan.

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c.

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization–tandem mass spectrometry, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

DETERGENT-SOLUBILIZED ESCHERICHIA COLI CYTOCHROME BO3 UBIQUINOL OXIDASE : A MONOMERIC, NOT A DIMERIC COMPLEX

The protein molecular weight, M r, and hydrodynamic radius, R s, of Triton X‐100‐solubilized Escherichia coli cytochrome bo 3 were evaluated by computer fitting of sedimentation velocity data with finite element solutions to the Lamm equation. Detergent‐solubilized cytochrome bo 3 sediments as a homogeneous species with an s 20,w of 6.75 s and a D 20,w of 3.71×10−7 cm2/s, corresponding to a R s of 5.8 nm and a M r of 144 000±3500. The protein molecular weight agrees very well with the value of 143 929 calculated from the four known subunit sequences and the value of 143 025 measured by MALDI mass spectrometry for the histidine‐tagged enzyme. We conclude that detergent‐solubilized E. coli ubiquinol oxidase is a monomeric complex of the four known subunits.

Inner mechanism of protection of mitochondrial electron-transfer proteins against oxidative damage. Focus on hydrogen peroxide decomposition

It is generally recognized that the mitochondria are the major source of reactive oxygen species including hydrogen peroxide (H2O2). Although the local concentration of H2O2 near the electron-transfer chain is potentially quite high, the chains components are rarely found to be significantly damaged by H2O2. Our experimental data, as well as the data published by others, suggest that mitochondrial electron-transfer proteins, which are in the first line to be harmed by ROS, are well prepared to defend themselves. One of such protection mechanism involves peroxidase/catalase-like activity of all major mitochondrial respiration chain players, which catalyze the decomposition of H2O2. Understanding the molecular mechanisms, by which mitochondrial electron-transfer proteins might defend themselves against an oxidative stress and therefore being a part of the mitochondrial antioxidant system, can help to clarify many controversial experimental data.

Role of cardiolipin in stability of integral membrane proteins

Cardiolipin (CL) is a unique phospholipid with a dimeric structure having four acyl chains and two phosphate groups found almost exclusively in certain membranes of bacteria and of mitochondria of eukaryotes. CL interacts with numerous proteins and has been implicated in function and stabilization of several integral membrane proteins (IMPs). While both functional and stabilization roles of CL in IMPs has been generally acknowledged, there are, in fact, only limited number of quantitative analysis that support this function of CL. This is likely caused by relatively complex determination of parameters characterizing stability of IMPs and particularly intricate assessment of role of specific phospholipids such as CL in IMPs stability. This review aims to summarize quantitative findings regarding stabilization role of CL in IMPs reported up to now.

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Cytochrome bc(1) isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc(1) is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc(1) was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc(1) retained nearly full activity, absorption spectra, subunit, and phospholipid composition.