Cytochrome c Oxidase (CCO)
Molecular oxygen (O2) functions primarily as a terminal acceptor of electrons on mitochondria and is consumed through this enzyme to generate H2O. This reaction is coupled with active transport of protons from the inner to the outer side of mitochondrial inner membrane. Reflux of the electron across the membrane through ATP synthase is necessary to maintain oxidative phosphorylation. Considering that more than 95 percent of oxygen consumed in our body is used for this reaction, it is not unreasonable to hypothesize that the enzyme per se serves as an oxygen sensor. Experiments using the photochemical action spectrum by Wilson et al.  first suggested that CCO serves as an oxygen sensor in the carotid body. The hypoxia-induced activation of the afferent neural burst of glomus cells in the body was CO sensitive, suggesting that the sensor is a heme protein, and the sensitivity profile of the CO effect to monochromatic light followed the absorption spectrum of CCO as a function of the wavelength of the light. However, mechanisms by which the CCO changes transfer the hypoxic signal to glomus cells are quite unknown in these experiments. Hypoxia-induced alterations in the CCO reaction trigger a number of events leading to remodeling of cell functions. Active transport of protons across the membrane is automatically reduced. NADH in the matrix is increased to inhibit dehydrogenase reactions in the Krebs cycle; this event secondarily decreases ATP and increases AMP to accelerate anaerobic glycolysis to compensate ATP synthesis. Cessation of oxygen consumption and accumulation of NADH causes generation of reactive oxygen species from the midpoint of the mitochondrial electron transfer system. In addition, a reduction of ATP synthesis inversely increases adenosine, a potent vasodilator. At present, it is unknown which of these events could play a critical role in transducing the hypoxic signal toward critical intracellular components to regulate vascular functions.
Oxygenases and Hemoglobin as a Class of Oxygen Sensors
Oxygenases constitute another class of oxygen sensors, since some of these enzymes accept molecular oxygen as a substrate and generate biologically active compounds as messenger molecules. Cyclooxygenases, cytochromes P450, NO synthase (NOS), and heme oxygenase (HO) are such candidate oxygenases that produce potent vasoactive mediators for hypoxic remodeling. NOS has largely been examined for its roles in regulation of neurovascular systems, but few studies showing its role in oxygen sensing have been available. Although a reduction of molecular oxygen in the cells could decrease the generation of the gas from the enzyme, it is unlikely that hypoxia causes a decrease in NO, since the half-life of NO by itself in vivo greatly depends on local oxygen tension . Recent studies suggest that not only endothelial cells but also erythrocyte-dependent recycling mechanisms for NO serve as resources of the gas available in microcirculation. Under hypoxic conditions, erythrocytes alter their hemoglobin allostery toward the T-state, and thereby increase the conductance of band III on their membranes; this reaction causes an increase in outflow of NO-glutathione into circulation . Such sequential reactions in and around the erythrocyte membrane also trigger the release of ATP which could secondarily stimulates NO in endothelial cells . Besides endothelial and neural NOS in situ, these circulating resources of NO or NO stimulators could explain an endothelial cell-independent fraction of NO supplied into microvascular beds in vivo .
Prabhakar et al.  suggested that HO-2 serves as an oxygen sensor in the carotid body; CO constitutively generated by HO-2 in glomus cells is decreased upon hypoxia and secondarily causes depolarization of the cells, leading to neural burst of their dopaminergic fibers. Interestingly, NOS and HO can be induced upon hypoxia as a result of tran-scriptional upregulation of the inducible enzymes, and compensate blood supply in hypoxic regions. Such events are mediated by nuclear translocation of hypoxia-inducible factor (HIF-1) upon hypoxia as described next.
Hypoxia-Inducible Factor (HIF)-1a and HIF Prolyl-hydroxylase
In the early 1990s, a novel hypoxia-inducible transcriptional factor, HIF-1, was identified. This factor binds at the 3' region of the hypoxia response element (HRE) of the erythropoietin gene after exposure to hypoxia . HIF-1 is a heterodimeric transcription factor that consists of two distinct components, the oxygen-sensitive a subunit (HIF-1a) and constitutively expressed b subunit that is also known as the aryl hydrocarbon receptor nuclear transloca-tor. Genes regulated by this transcription factor involve erythropoietin, transferrin, cyclin E, VEGF, inducible NO synthase, heme oxygenase-1, and a series of glycolytic enzymes.
Until recently, mechanisms by which organisms sense alterations in oxygen concentration and subsequently induce HIF-1 activities remained unknown. Under normoxia, HIF-1a binds to a von Hippel-Lindau tumor suppressor gene product, pVHL, that rapidly leads to ubiquitin-dependent proteolysis of HIF-1 a . On the other hand, HIF-1 a does not bind to pVHL and is rapidly accumulated in nuclei within a few minutes, suggesting structural alterations in HIF-1 a upon hypoxia. Two distinct molecular mechanisms by which HIF-1 a alters its structure to escape from the pVHL binding involve oxygen-dependent hydroxylation of specific amino acid residues. First, hydroxylation of prolyl residues of the protein (Pro402 and Pro564) is necessary to escape from pVHL-dependent ubiquitination and requires a prolyl-4-hydroxylase, a member of a subfamily of novel protein hydroxylases distinct from those for collagen stabilization. In mammals, three homologs termed HPH-1, -2, and -3 have been cloned, constituting the superfamily of dioxygenases, and require molecular oxygen and 2-oxoglu-tarate as cosubstrates. Second, hydroxylation of a critical asparagine residue (Asn803 in HIF-1 a) occurs in an oxygen-dependent manner and in turn renders it unable to associate with CBP/p300 transcriptional coactivators. Lando and colleagues  determined that the asparagine hydroxylase involved in the catalytic reaction is identical to factor-inhibiting hypoxia-inducible factor-1 (FIH-1), which was initially identified by Semenza and coworkers as a novel HIF-1-binding protein. This enzyme is a 2-oxoglu-tarate-dependent dioxygenase that utilizes molecular oxygen to modify its substrate. Through these mechanisms, oxygen tension not only affects HIF-1 a degradation but also dictates its subcellular localization.
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