Rabia Ramzan

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation.

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Phosphorylation and Kinetics of Mammalian Cytochrome c Oxidase

The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.

New extension of the Mitchell Theory for oxidative phosphorylation in mitochondria of living organisms.

The Mitchell Theory implies the proton motive force Deltap across the inner mitochondrial membrane as the energy-rich intermediate of oxidative phosphorylation. Deltap is composed mainly of an electrical (DeltaPsi(m)) and a chemical part (DeltapH) and generated by the respiratory chain complexes I, III and IV. It is consumed mostly by the ATP synthase (complex V) to produce ATP. The free energy of electron transport within the proton pumps is sufficient to generate Deltap of about 240 mV. The proton permeability of biological membranes, however, increases exponentially above 130 mV leading to a waste of energy at high values (DeltaPsi(m)>140 mV). In addition, at DeltaPsi(m)>140 mV, the production of the superoxide radical anion O(2)(-) at complexes I, II and III increases exponentially with increasing DeltaPsi(m). O(2)(-) and its neutral product H(2)O(2) (=ROS, reactive oxygen species) induce oxidative stress which participates in aging and in the generation of degenerative diseases. Here we describe a new mechanism which acts independently of the Mitchell Theory and keeps DeltaPsi(m) at low values through feedback inhibition of complex IV (cytochrome c oxidase) at high ATP/ADP ratios, thus preventing the formation of ROS and maintaining high efficiency of oxidative phosphorylation.

Degenerative diseases, oxidative stress and cytochrome c oxidase function

Aging and degenerative diseases are associated with increased levels of reactive oxygen species (ROS). ROS are mostly produced in mitochondria, and their levels increase with higher mitochondrial membrane potential. Cellular respiratory control is based on inhibition of respiration by high membrane potentials. However, we have described a second mechanism of respiratory control based on allosteric inhibition of cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, at high ATP:ADP ratios. The mechanism is independent of membrane potential. We have proposed that feedback inhibition of CcO by ATP keeps the membrane potential and ROS production at low levels. Various forms of stress switch off allosteric ATP-inhibition via reversible dephosphorylation of CcO, resulting in increased membrane potential and cellular ROS levels. This mechanism is proposed to represent a missing molecular link between stress and degenerative diseases.

The role of mitochondrial membrane potential in ischemic heart failure.

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ(m)). The low ΔΨ(m) values of the normal beating heart (100-140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ(m) values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.

Multiple phosphorylations of cytochrome c oxidase and their functions

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is regulated by isozyme expression, allosteric effectors such as the ATP/ADP ratio, and reversible phosphorylation. Of particular interest is the “allosteric ATP‐inhibition,” which has been hypothesized to keep the mitochondrial membrane potential at low healthy values (<140 mV), thus preventing the formation of superoxide radical anions, which have been implicated in multiple degenerative diseases. It has been proposed that the “allosteric ATP‐inhibition” is switched on by the protein kinase A‐dependent phosphorylation of COX. The goal of this study was to identify the phosphorylation site(s) involved in the “allosteric ATP‐inhibition” of COX. We report the mass spectrometric identification of four new phosphorylation sites in bovine heart COX. The identified phosphorylation sites include Tyr‐218 in subunit II, Ser‐1 in subunit Va, Ser‐2 in subunit Vb, and Ser‐1 in subunit VIIc. With the exception of Ser‐2 in subunit Vb, the identified phosphorylation sites were found in enzyme samples with and without “allosteric ATP inhibition,” making Ser‐2 of subunit Vb a candidate site enabling allosteric regulation. We therefore hypothesize that additional phosphorylation(s) may be required for the “allosteric ATP‐inhibition,” and that these sites may be easily dephosphorylated or difficult to identify by mass spectrometry.

Individual Biochemical Behaviour Versus Biological Robustness: Spotlight on the Regulation of Cytochrome c Oxidase

During evolution from prokaryotes to eukaryotes, the main function of cytochrome c oxidase (COX), i.e., the coupling of oxygen reduction to proton translocation without the production of ROS (reactive oxygen species) remained unchanged demonstrating its robustness. A new regulation of respiration by the ATP/ADP ratio was introduced in eukaryotes based on nucleotide interaction with the added COX subunit IV. This allosteric ATP-inhibition was proposed to keep the mitochondrial membrane potential (ΔΨ(m)) at low healthy values and thus prevents the formation of ROS at complexes I and III. ROS have been implicated in various degenerative diseases. The allosteric ATP-inhibition of COX is reversibly switched on and off by phosphorylation of COX at a serine or threonine. In more than 100 individual preparations of rat heart and liver mitochondria, prepared under identical conditions, the extent of allosteric ATP-inhibition varied. This variability correlates with the variable inhibition of uncoupled respiration in intact isolated mitochondria by ATP. It is concluded that in higher organisms the allosteric ATP-inhibition is continually switched on and off by neuronal signalling in order to change oxidative phosphorylation from optimal efficiency with lower rate of ATP synthesis under resting conditions (low ΔΨ(m) and ROS production) to maximal rate of ATP synthesis under active (working, stress) conditions (elevated ΔΨ(m) and ROS production).

GAPDH: the missing link between glycolysis and mitochondrial oxidative phosphorylation?

The main function of glycolysis and oxidative phosphorylation is to produce cellular energy in the form of ATP. In the present paper we propose a link between both of these energy-regulatory processes in the form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CytOx (cytochrome c oxidase). GAPDH is the sixth enzyme of glycolysis, whereas CytOx is the fourth complex of the mitochondrial oxidative phosphorylation system. In MS analysis, GAPDH was found to be associated with a BN-PAGE (blue native PAGE)-isolated complex of CytOx from bovine heart tissue homogenates. Both GAPDH and CytOx are highly regulated under normal energy metabolic conditions, but both of these enzymes are highly deregulated in the presence of oxidative stress. The interaction of GAPDH with CytOx could be the point of interest as it has already been shown that GAPDH protein damage results in a marked decrease in cellular ATP levels. On the other hand, decreasing the ATP/ADP ratio may ultimately result in switching off the allosteric ATP inhibition of CytOx leading to increased ROS (reactive oxygen species), cytochrome c release and apoptosis. Moreover, we have previously reported that allosteric ATP inhibition of CytOx is responsible for keeping the membrane potential at low healthy values, thus avoiding the production of ROS and this allosteric ATP inhibition is switched on at a high ATP/ADP ratio. So, in the present paper, we propose a scheme that could prove to be a link between these two enzymes and their role in the prevalence of diseases.

Discovering the phosphoproteome of the hydrophobic cytochrome c oxidase membrane protein complex.

Many cellular processes are regulated by reversible phosphorylation to change the activity state of proteins. One example is cytochrome c oxidase (COX) with its important function for energy metabolism in the mitochondria. The phosphorylation of this enzyme is a prerequisite for the allosteric ATP-inhibition and therefore necessary to adapt energy production to ATP demand of the cell. Its hydrophobic nature hampers the recognition of phosphorylated amino acids in most subunits of this complex, and as a consequence, only a few phosphorylation sites were identified by mass spectrometry. We describe here a method that enables the analysis of integral membrane proteins by chemical cleavage with cyanogen bromide (BrCN), a method that improves the mass spectrometric detection of hydrophobic proteins. The low abundance of phosphopeptides requires efficient enrichment techniques, such as TiO(2)-based methods. However, this strategy failed in our hands when just BrCN-cleaved peptides were used. Only an additional size-reduction with trypsin produced peptides with optimal properties for enrichment and MS-identification. Another bottleneck was the correct assignment of phosphoserine and phosphothreonine because peptide-ion fragmentation by collision induced dissociation (CID) often results in neutral loss of HPO(3) or H(2)PO(4) from the precursor, decreasing fragmentations that define the peptide sequence and the phosphorylation site. The additional usage of electron transfer dissociation (ETD) as an alternative fragmentation method enabled the precise assignment of the phosphorylated amino acids. In a total of six, new phosphorylation sites of four COX-subunits were identified by this strategy.

Heat shock protein expression and change of cytochrome c oxidase activity: presence of two phylogenic old systems to protect tissues in ischemia and reperfusion

Induction of heat shock proteins (hsp) has been shown to protect cells from ischemia by providing transient tolerance against myocardial injury and improving postischemic functional recovery. Attenuation of ATP depletion and earlier restoration of ATP content on reperfusion are thought to play a role in this scenario. Hsp induction is accompanied by altered enzyme activity of the respiratory chain, the major generator of ATP under physiological conditions. This report addresses the question whether processing and final assembly of the active holoenzyme cytochrome c oxidase (CcO, complex IV), member of the respiratory chain, is compromised under hypoxic conditions unless protected by stress proteins. Special focus is laid on function of the enzyme’s subunits and importance of cellular energy availability and maintenance.