Nitric oxide produced by cytochrome c oxidase helps stabilize HIF-1α in hypoxic mammalian cells.

The mitochondrial respiratory chain has been reported to play a role in the stabilization of HIF-1α when mammalian cells experience hypoxia, most likely through the generation of free radicals. Although previous studies have suggested the involvement of superoxide catalyzed by complex III more recent studies raise the possibility that nitric oxide (NO) catalyzed by cytochrome c oxidase (Cco/NO), which functions in hypoxic signaling in yeast, may also be involved. Herein, we have found that HEK293 cells, which do not express a NOS isoform, possess Cco/NO activity and that this activity is responsible for an increase in intracellular NO levels when these cells are exposed to hypoxia. By using PTIO, a NO scavenger, we have also found that the increased NO levels in hypoxic HEK293 cells help stabilize HIF-1α. These findings suggest a new mechanism for mitochondrial involvement in hypoxic signaling in mammalian cells.

Identification of potential calorie restriction-mimicking yeast mutants with increased mitochondrial respiratory chain and nitric oxide levels.

Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Therapeutic photobiomodulation: nitric oxide and a novel function of mitochondrial cytochrome c oxidase.

Currently, light therapies are widely used in both human and veterinarian medicine. The application of light to clinical therapeutics includes: photodynamic therapy, used to kill cancer cells; UVA therapies, used to treat a variety of skin diseases; and photobiomodulation, used to promote cell growth and recovery from injury. Photobiomodu-lation uses light emitting diodes (LEDs) or low energy lasers, which emit light in the visible red to near infrared range. Light in this range penetrates tissue reasonably well, lacks the carcinogenic/mutagenic properties of UV light, and acts on an endogenous photoreceptor which likely acts to initiate light-altered signaling pathways. Although early studies identified mitochondrial cytochrome c oxidase as an endogenous photoreceptor for photobiomodulation, the cellular and molecular mechanisms underlying photobiomodulation have not been clear. Three recent findings provide important new insight. First, nitric oxide has been implicated. Second, cytochrome c oxidase, an enzyme known to reduce oxygen to water at the end of the mitochondrial respiratory chain, has been shown to have a new enzymatic activity--the reduction of nitrite to nitric oxide. This nitrite reductase activity is elevated under hypoxic conditions but also occurs under normoxia. And third, low intensity light enhances nitric oxide synthesis by cytochrome c oxidase without altering its ability to reduce oxygen. From these findings, we propose that cytochrome c oxidase functions in photobiomodulation by producing nitric oxide, a signaling molecule which can then function in both intra- and extracellular signaling pathways. We also propose that the effectiveness of photobiomodulation is under the control of tissue oxygen and nitrite levels.

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.

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 (NO(2) (-))-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.

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.

The absence of a mitochondrial genome in rho0 yeast cells extends lifespan independently of retrograde regulation.

The absence of mtDNA in rho0 yeast cells affects both respiration and mitochondrial-nuclear communication (e.g., retrograde regulation, intergenomic signaling, or pleiotropic drug resistance). Previously, it has been reported that some rho0 strains have increased replicative lifespans, attributable to the lack of respiration and retrograde regulation. Here, we have been able to confirm that rho0 cells exhibit increased replicative lifespans but have found that this is not associated with the lack of respiration or reduced oxidative stress but instead, is related to the lack of mtDNA per se in rho0 cells. Also, we find no correlation between the strength of retrograde regulation and lifespan. Furthermore, we find that pdr3- or rtg2- mutations are not responsible for lifespan extension in rho0 cells, ruling out a specific role for PDR3-pleiotropic drug resistance or RGT2-retrograde regulation pathways in the extended lifespans of rho0 cells. Surprisingly, Rtg3p, which acts downstream of Rtg2p, is required for lifespan increase in rho0 cells. Together, these findings indicate that the loss of mtDNA per se and not the lack of respiration lead to extended longevity in rho0 cells. They also suggest that Rtg3p, acting independently of retrograde regulation, mediates this effect, possibly via intergenomic signaling.

Multiple pathways of mitochondrial-nuclear communication in yeast: intergenomic signaling involves ABF1 and affects a different set of genes than retrograde regulation.

Mitochondrial-nuclear communication is taking on increased importance in models of oxygen sensing, oxidative stress, aging, and disease. The deletion of the mitochondrial genome (mtDNA) and, hence, the ability to respire, affects expression of several nuclear genes through at least two different mitochondrial-nuclear communication pathways. One of the pathways, retrograde regulation, is activated by a reduction in respiration, while another, intergenomic signaling, is unaffected by respiration but requires mtDNA. Using DNA microarrays, we identify here a set of nuclear genes in Saccharomyces cerevisiae that are targets of intergenomic signaling. These nuclear genes are down-regulated in rho degrees cells that lack mtDNA but not in nuclear pet mutant rho(+)cells that possess mtDNA but lack respiration. Many of these nuclear genes encode mitochondrial proteins, implying that intergenomic signaling functions in coordinating mitochondrial and nuclear gene expression. In addition, analyses of deletion and linker scanning mutations in the promoter of the COX6 gene, a nuclear gene affected by intergenomic signaling, suggest an involvement of Abf1p transcription factor in intergenomic signaling. Together, these findings indicate that intergenomic signaling is distinct from retrograde regulation both in the nuclear genes that it regulates and in the way in which it affects their expression.

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 (NO(2)(-))-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 NO(2)(-)-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 NO(2)(-)-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.

Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes.

Eukaryotic cells respond to low-oxygen concentrations by upregulating hypoxic nuclear genes (hypoxic signaling). Although it has been shown previously that the mitochondrial respiratory chain is required for hypoxic signaling, its underlying role in this process has been unclear. Here, we find that yeast and rat liver mitochondria produce nitric oxide (NO) at dissolved oxygen concentrations below 20 microM. This NO production is nitrite (NO2-) dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. Mitochondrial NO production in yeast is influenced by the YHb flavohemoglobin NO oxidoreductase, stimulates expression of the hypoxic nuclear gene CYC7, and is accompanied by an increase in protein tyrosine nitration. These findings demonstrate an alternative role for the mitochondrial respiratory chain under hypoxic or anoxic conditions and suggest that mitochondrially produced NO is involved in hypoxic signaling, possibly via a pathway that involves protein tyrosine nitration.

Effects of a transition from normoxia to anoxia on yeast cytochrome c oxidase and the mitochondrial respiratory chain: implications for hypoxic gene induction.

Previous studies have demonstrated that the mitochondrial respiratory chain and cytochrome c oxidase participate in oxygen sensing and the induction of some hypoxic nuclear genes in eukaryotes. In addition, it has been proposed that mitochondrially-generated reactive oxygen and nitrogen species function as signals in a signaling pathway for the induction of hypoxic genes. To gain insight concerning this pathway, we have looked at changes in the functionality of the yeast respiratory chain as cells experience a shift from normoxia to anoxia. These studies have revealed that yeast cells retain the ability to respire at normoxic levels for up to 4 h after a shift and that the mitochondrial cytochrome levels drop rapidly to 30--50% of their normoxic levels and the turnover rate of cytochrome c oxidase (COX) increases during this shift. The increase in COX turnover rate cannot be explained by replacing the aerobic isoform, Va, of cytochrome c oxidase subunit V with the more active hypoxic isoform, Vb. We have also found that mitochondria retain the ability to respire, albeit at reduced levels, in anoxic cells, indicating that yeast cells maintain a functional mitochondrial respiratory chain in the absence of oxygen. This raises the intriguing possibility that the mitochondrial respiratory chain has a previously unexplored role in anoxic cells and may function with an alternative electron acceptor when oxygen is unavailable.

Yeast flavohemoglobin, a nitric oxide oxidoreductase, is located in both the cytosol and the mitochondrial matrix: effects of respiration, anoxia, and the mitochondrial genome on its intracellular level and distribution.

Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in both the oxidative and nitrosative stress responses in Saccharomyces cerevisiae. Previous studies have shown that the expression of YHB1 is optimal under normoxic or hyperoxic conditions, yet respiring yeast cells have low levels of reduced YHb pigment as detected by carbon monoxide (CO) photolysis difference spectroscopy of glucose-reduced cells. Here, we have addressed this apparent discrepancy by determining the intracellular location of the YHb protein and analyzing the relationships between respiration, YHb level, and intracellular location. We have found that although intact respiration-proficient cells lack a YHb CO spectral signature, cell extracts from these cells have both a YHb CO spectral signature and nitric oxide (NO) consuming activity. This suggests either that YHb cannot be reduced in vivo or that YHb heme is maintained in an oxidized state in respiring cells. By using an anti-YHb antibody and CO difference spectroscopy and by measuring NO consumption, we have found that YHb localizes to two distinct intracellular compartments in respiring cells, the mitochondrial matrix and the cytosol. Moreover, we have found that the distribution of YHb between these two compartments is affected by the presence or absence of oxygen and by the mitochondrial genome. The findings suggest that YHb functions in oxidative stress indirectly by consuming NO, which inhibits mitochondrial respiration and leads to enhanced production of reactive oxygen species, and that cells can regulate intracellular distribution of YHb in accordance with this function.

A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant.

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.

Mitochondrial protein oxidation in yeast mutants lacking manganese-(MnSOD) or copper- and zinc-containing superoxide dismutase (CuZnSOD): evidence that MnSOD and CuZnSOD have both unique and overlapping functions in protecting mitochondrial proteins from oxidative damage.

Saccharomyces cerevisiae expresses two forms of superoxide dismutase (SOD): MnSOD, encoded by SOD2, which is located within the mitochondrial matrix, and CuZnSOD, encoded by SOD1, which is located in both the cytosol and the mitochondrial intermembrane space. Because two different SOD enzymes are located in the mitochondrion, we examined the relative roles of each in protecting mitochondria against oxidative stress. Using protein carbonylation as a measure of oxidative stress, we have found no correlation between overall levels of respiration and the level of oxidative mitochondrial protein damage in either wild type or sod mutant strains. Moreover, mitochondrial protein carbonylation levels in sod1, sod2, and sod1sod2 mutants are not elevated in cells harvested from mid-logarithmic and early stationary phases, suggesting that neither MnSOD nor CuZnSOD is required for protecting the majority of mitochondrial proteins from oxidative damage during these early phases of growth. During late stationary phase, mitochondrial protein carbonylation increases in all strains, particularly in sod1 and sod1sod2 mutants. By using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we have found that specific proteins become carbonylated in sod1 and sod2 mutants. We identified six mitochondrial protein spots representing five unique proteins that become carbonylated in a sod1 mutant and 19 mitochondrial protein spots representing 11 unique proteins that become carbonylated in a sod2 mutant. Although some of the same proteins are carbonylated in both mutants, other proteins are not. These findings indicate that MnSOD and CuZnSOD have both unique and overlapping functions in the mitochondrion.

A mechanism of oxygen sensing in yeast. Multiple oxygen-responsive steps in the heme biosynthetic pathway affect Hap1 activity.

Heme plays central roles in oxygen sensing and utilization in many living organisms. In yeast, heme mediates the effect of oxygen on the expression of many genes involved in using or detoxifying oxygen. However, a direct link between intracellular heme level and oxygen concentration has not been vigorously established. In this report, we have examined the relationships among oxygen levels, heme levels, Hap1 activity, and HAP1 expression. We found that Hap1 activity is controlled in vivo by heme and not by its precursors and that heme activates Hap1 even in anoxic cells. We also found that Hap1 activity exhibits the same oxygen dose-response curves as Hap1-dependent aerobic genes and that these dose-response curves have a sharp break at approximately 1 microM O2. The results show that the intracellular signaling heme level, reflected as Hap1 activity, is closely correlated with oxygen concentration. Furthermore, we found that bypass of all heme synthetic steps but ferrochelatase by deuteroporphyrin IX does not circumvent the need for oxygen in Hap1 full activation by heme, suggesting that the last step of heme synthesis, catalyzed by ferrochelatase, is also subjected to oxygen control. Our results show that multiple heme synthetic steps can sense oxygen concentration and provide significant insights into the mechanism of oxygen sensing in yeast.

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-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-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-H(2)-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.