Robert O. Poyton

Mitochondrial generation of free radicals and hypoxic signaling

Most reactive oxygen species (ROS) are generated in cells by the mitochondrial respiratory chain. Mitochondrial ROS production is modulated largely by the rate of electron flow through respiratory chain complexes. Recently, it has become clear that under hypoxic conditions, the mitochondrial respiratory chain also produces nitric oxide (NO), which can generate other reactive nitrogen species (RNS). Although excess ROS and RNS can lead to oxidative and nitrosative stress, moderate to low levels of both function in cellular signaling pathways. Especially important are the roles of these mitochondrially generated free radicals in hypoxic signaling pathways, which have important implications for cancer, inflammation and a variety of other diseases.

Exposure of Yeast Cells to Anoxia Induces Transient Oxidative Stress IMPLICATIONS FOR THE INDUCTION OF HYPOXIC GENES

The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes. Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction. Recent studies with mammalian cells have produced conflicting results concerning this question. These studies have relied almost exclusively on fluorescent dyes to measure ROS levels. Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g. the accumulation of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stress-induced gene, e.g. SOD1. Here we used these approaches as well as a fluorescent dye, carboxy-H2-dichloro-dihydrofluorescein diacetate (carboxy-H2-DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia. These studies also reveal that carboxy-H2-DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress. The mitochondrial respiratory chain is responsible for much of this carbonylation. Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes.

Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling

Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (NO2−)-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the NO2−-dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on NO2−-dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells.

Expression and function of cytochrome c oxidase subunit isologues. Modulators of cellular energy production

Attempts to understand the regulation of cellular energy production in nonphotosynthetic eukaryotic cells have focused on the components of the mitochondrial respiratory chain and its three sites of energy conservation. Although a number of factors could interact to affect the rate of mitochondrial respiration and oxidative phosphorylation, recent studies have led to the proposal that cytochrome c oxidase is a key enzyme in the overall regulation of cellular energy production in eukaryotes.’.’ This hypothesis is supported by a number of findings. First, the redox reactions between NADH and cytochrome c in the mitochondrial respiratory chain (i.e., those that include the first two sites of oxidative phosphorylation) are near equilibrium3-” whereas the redox reactions between cytochrome c and 0,, which are catalyzed by cytochrome c oxidase, are essentially irreversible.’ Second, the application of control to oxidative phosphorylation has revealed that cytochrome c oxidase is one of two major steps that have significant “control strength” in mitochondria from both higher eukaryotes (e.g. rat liverf4) and lower eukaryotes (e.g. Saccharomyces cerevisiae”). Third, cytochromes aa, can be limiting in amount with respect to other respiratory chain components in some organisms (e.g., Candida parapsilosis, 5‘. cerevisiaeI6) and tissues (e.g. bovine liver”). Fourth, ATP (and other anions) has a marked effect on the kinetic properties of cytochrome c oxidase from a variety of eukaryotes”-” by binding to subunits that are thought not to carry the redox centers of the holoenzyme.22 Together these findings suggest that one, or more, of the reactions catalyzed by cytochrome c oxidase is equivalent to the committed step in a metabolic pathway. As such, it is an important control point that somehow matches the level of respiration and oxidative phosphorylation to cellular energy requirements. At present, it is unclear how eukaryotic cells alter their cytochrome c oxidase activity levels in response to changes in energy demand. In principle, two general types of regulation are possible: short term and long term. These two types of regulation are distinguishable by response time and by a requirement for protein synthesis and/or turnover. Short-term regulation is immediate, could be affected by allosteric regulation (via ATP and/or other metabolitesz3), and does not require protein synthesis or turnover. Long-term regulation would be affected by changing the number of

FUNCTION AND EXPRESSION OF FLAVOHEMOGLOBIN IN SACCHAROMYCES CEREVISIAE : EVIDENCE FOR A ROLE IN THE OXIDATIVE STRESS RESPONSE

We have studied the function and expression of the flavohemoglobin (YHb) in the yeast Saccharomyces cerevisiae. This protein is a member of a family of flavohemoproteins, which contain both heme and flavin binding domains and which are capable of transferring electrons from NADPH to heme iron. Normally, actively respiring yeast cells have very low levels of the flavohemoglobin. However, its intracellular levels are greatly increased in cells in which the mitochondrial electron transport chain has been compromised by either mutation or inhibitors of respiration. The expression of the flavohemoglobin gene, YHB1, of S. cerevisiae is sensitive to oxygen. Expression is optimal in hyperoxic conditions or in air and is reduced under hypoxic and anaerobic conditions. The expression of YHB1 in aerobic cells is enhanced in the presence of antimycin A, in thiol oxidants, or in strains that lack superoxide dismutase. All three conditions lead to the accumulation of reactive oxygen species and promote oxidative stress. To study the function of flavohemoglobin in vivo, we created a null mutation in the chromosomal copy of YHB1. The deletion of the flavohemoglobin gene in these cells does not affect growth in either rho° or rho+ genetic backgrounds. In addition, a rho+ strain carrying a yhb1− deletion has normal levels of both cyanide-sensitive and cyanide-insensitive respiration, indicating that the flavohemoglobin does not function as a terminal oxidase and is not required for the function or expression of the alternative oxidase system in S. cerevisiae. Cells that carry a yhb1−deletion are sensitive to conditions that promote oxidative stress. This finding is consistent with the observation that conditions that promote oxidative stress also enhance expression of YHB1. Together, these findings suggest that YHb plays a role in the oxidative stress response in yeast.

Mitochondria and hypoxic signaling: a new view.

Eukaryotic cells respond to low oxygen concentrations by upregulating hypoxic and downregulating aerobic nuclear genes (hypoxic signaling). Most of the oxygen‐regulated genes in yeast require the mitochondrial respiratory chain for their up‐ or downregulation when cells experience hypoxia. Although it was shown previously that the mitochondrial respiratory chain is required for the upregulation of some hypoxic genes in both yeast and mammalian cells, its underlying role in this process has been unclear. Recently, we have reported that mitochondria produce nitric oxide (NO•) when oxygen becomes limiting. This NO• production is nitrite (NO2−)‐dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH‐dependent fashion. We call this activity Cco/NO• and incorporate it into a new model for hypoxic signaling. In addition, we have found that some of the NO• produced by Cco/NO• is released from cells, raising the possibility that mitochondrially generated NO• also functions in extracellular hypoxic signaling pathways.

Models for oxygen sensing in yeast: Implications for oxygen-regulated gene expression in higher eucaryotes

Adaptation to changes in oxygen tension in cells, tissues, and organisms depends on changes in the level of expression of a large and diverse set of proteins. It is likely that most cells and tissues possess an oxygen sensing apparatus and signal transduction pathways for regulating expression of oxygen-responsive genes. Although progress has been made in understanding the transcriptional machinery involved in oxygen-regulated gene expression of eucaryotic genes the underlying mechanism(s) of oxygen sensing and the signaling pathways that connect oxygen sensor(s) to the transcription machinery of eucaryotes are still poorly understood. The yeast Saccharomyces cerevisiae is ideal for addressing these problems. Indeed, it is well-suited for broadly based studies on oxygen sensing at the cellular level because it lends itself well to genetic and biochemical studies and because its genome has been completely sequenced. This review focuses on oxygen-regulated gene expression and current models for oxygen sensing in this yeast and then considers their applicability for understanding oxygen sensing in mammals.

Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes.

In Saccharomyces cerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension.

Effects of Anoxia and the Mitochondrion on Expression of Aerobic Nuclear COX Genes in Yeast EVIDENCE FOR A SIGNALING PATHWAY FROM THE MITOCHONDRIAL GENOME TO THE NUCLEUS

Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of somehypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeastaerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclearcox mutants and mitochondrial rho 0and mit − mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclearCOX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.

Low intensity light stimulates nitrite-dependent nitric oxide synthesis but not oxygen consumption by cytochrome c oxidase: Implications for phototherapy

Cytochrome c oxidase (Cco) has been reported to be a receptor for some of the beneficial effects of low intensity visible and near-infrared light on cells and tissues. Here, we have explored the role of low intensity light in affecting a newly described function of Cco, its ability to catalyze nitrite-dependent nitric oxide (NO) synthesis (Cco/NO). Using a new assay for Cco/NO we have found that both yeast and mouse brain mitochondrial Cco produce NO over a wide range of oxygen concentrations and that the rate of NO synthesis increases as the oxygen concentration decreases, becoming optimal under hypoxic conditions. Low intensity broad-spectrum light increases Cco/NO activity in an intensity-dependent fashion but has no effect on oxygen consumption by Cco. By using a series of bandpass filters and light emitting devices (LEDs) we have determined that maximal stimulation of Cco/NO activity is achieved by exposure to light whose central wavelength is 590 ± 14 nm. This wavelength of light stimulates Cco/NO synthesis at physiological nitrite concentrations. These findings raise the interesting possibility that low intensity light exerts a beneficial effect on cells and tissues by increasing NO synthesis catalyzed by Cco and offer a new explanation for the increase in NO bioavailability experienced by tissue exposed to light.