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Symposium Reports |
1 Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
Abstract
Myoglobin and haemoglobin, the respiratory pigments of mammals and some molluscs, annelids and arthropods, belong to an ancient superfamily of haem-associated globin proteins. Members of this family share common structural and spectral features. They also share some general functional characteristics, such as the ability to bind ligands, e.g. O2, CO and NO, at the iron atom and to undergo redox changes. These properties are used in vivo to perform a wide range of biochemical and physiological roles. While it is acknowledged that the major role of haemoglobin is to bind oxygen reversibly and deliver it to the tissues, this is not its only function, while the often-stated role of myoglobin as an oxygen storage protein is possibly a misconception. Furthermore, haemoglobin and myoglobin express enzymic activities that are important to their function, e.g. NO dioxygenase activity or peroxidatic activity that may be partly responsible for pathophysiology following haemorrhage. Evidence for these functions is described, and the discussion extended to include proteins that have recently been discovered and that are expressed at low levels within the cell. These proteins are hexaco-ordinate, unlike haemoglobin and myoglobin, and are widely distributed throughout the animal kingdom (e.g. neuroglobins and cytoglobins). They may have specialist roles in oxygen delivery to particular sites within the cell but may also perform roles associated with O2 sensing and signalling and in responses to stress, e.g. protection from reactive oxygen and nitrogen species. Haemoglobins are also widespread in plants and bacteria and may serve similar protective functions.
(Received 26 July 2007;
accepted after revision 25 October 2007; first published online 2 November 2007)
Corresponding author M. T. Wilson: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Email: wilsmt{at}essex.ac.uk
The tetrapyrrole ring structure of the haem group provides four nitrogen ligands to the central iron atom that lies at the heart of all haemoproteins. This planar cofactor is buried within the hydrophobic interior of a protein (a globin in the cases discussed here) that also provides a further nitrogen ligand from a histidine residue (the proximal histidine). The sixth co-ordination site of the iron remains available to bind external ligands. When the iron is in the ferrous (FeII) redox state it can reversibly bind gaseous ligands such as oxygen (but also CO and NO), hence enabling the protein to perform the functional roles of oxygen storage (myoglobin, Mb, but see below) and transport (haemoglobin, Hb). The oxygen affinity can be tuned to meet the physiological requirements of the organism and, through allosteric interactions, made responsive to metabolites, pH, CO2, etc. These functions are well described elsewhere, and this review will deal with other important functions of the haem-containing globins that are less well recognized.
The enzymatic activity of myoglobin and haemoglobin under pathological conditions
The globular haem-containing proteins myoglobin and haemoglobin are two of the most studied and characterized proteins. While the function of Hb is clearly to carry oxygen from the lungs to tissues, our understanding of the physiological function of Mb has changed over the past decade. Previously, Mb has been considered an oxygen storage protein that augmented oxygen diffusion in high-oxygen-consuming, mitochondria-rich muscles (e.g. heart). Following the observation that mice lacking the gene to produce Mb had no obvious change in phenotype other than ‘white’ muscle (Godecke et al. 1999), the physiological role of Mb was reassessed, and it is now considered also to be an intracellular scavenger of nitric oxide, protecting NO-sensitive respiratory enzymes such as cytochrome c oxidase (Eich et al. 1996; Brunori, 2001), as follows:
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This enzymatic activity of Mb and Hb was believed to have no relevance in vivo. However, in the last 20 years it has become apparent that the redox chemistry of Hb and Mb leading to undesired oxidation reactions has real biological importance, typically under pathophysiological conditions following a haemolytic event or ischaemia–reperfusion injury. A cascade of oxidation reactions initiated by Mb or Hb can occur if the oxidant–antioxidant balance is disturbed through: (1) overwhelming of the antioxidant defences, e.g. during reperfusion following ischaemia; or (2) a lack of antioxidants, such as following a haemolytic event, when the haem proteins find themselves in unfamiliar environments with limited antioxidant concentrations. A haemolytic event, such as rhabdomyolysis (muscle breakdown caused by trauma, hyperthermia, hypothermia, alcohol or drug abuse, exercise, etc.) or subarachnoid haemorrhage, can contaminate the kidney or cerebrospinal fluid with haem protein, which leads to kidney dysfunction or delayed vasospasm, respectively. Isoprostanes, a series of potent vasoactive lipid oxidation products, have been linked with the pathogenesis of both kidney failure and delayed vasospasm in the brain (Moore et al. 1998; Sakamoto et al. 2002). It was initially believed that Fenton chemistry by free iron, derived from haem breakdown, was the only mechanism to initiate isoprostane formation through lipid oxidation. The evidence for this mechanism primarily results from the amelioration of clinical conditions from the use of iron chelators, such as desferrioxamine (Zager, 1992). However, evidence that supports the hypothesis that intact Mb and Hb may also act as initiators of oxidative stress in disease states includes the following.
(1) Myoglobin and Hb induce isoprostane formation from low-density lipoproteins in vitro (Moore et al. 1998).
(2) Breakdown of the haem moiety of Mb or Hb by haem oxygenase-1 is indispensable for ameliorating posthaemolytic disease states (Nath et al. 2000; Ono et al. 2002).
(3) Haem to protein cross-linked forms of Mb, a marker of the previous redox activity of Mb or Hb, have been identified in the urine of patients diagnosed with rhabdomyolysis and in the kidneys of animal models (Holt et al. 1999; Reeder et al. 2002a).
(4) Many iron chelators can act as electron-donating agents and are thus efficient reductants of ferryl haem, preventing lipid oxidation by intact haem proteins independent of their ability to chelate iron. These chelators also inhibit haem to protein cross-linking (Cooper et al. 1994; Reeder & Wilson, 2005; Merkofer et al. 2006).
There is now considerable evidence showing that Mb and Hb can act as rogue enzymes involved in redox chemistry in vivo. It is interesting to note that a treatment for rhabdomyolysis (with limited use owing to potentially serious side-effects) is alkalinization of the patient's blood/urine (Zager, 1989; Better & Stein, 1990). Increasing the pH of the environment of Mb and Hb slows down the redox chemistry by stabilizing the ferryl form and decreasing the rate of lipid oxidation (Moore et al. 1998). The protonated form of ferryl Mb or Hb (Mb/Hb(IV)-OH–) is much more reactive than the unprotonated form, being chemically equivalent to the ferric species plus a radical. Hence, although not technically a radical, it has radical-like properties that enhance the pro-oxidant nature of the haem protein at low pH values (Silaghi-Dumitrescu et al. 2007). Protonation of ferryl Mb or Hb through acidosis may be responsible for cytotoxicity and also induces the formation of haem to protein cross-linking (Reeder et al. 2002b). Cross-linked Mb is itself five times more cytotoxic than the native form, owing to enhanced peroxidase activities (Vuletich et al. 2000).
The haemoglobin superfamily: a series of redox active enzymes?
The pseudo-enzymatic activities of Mb and Hb, once believed only to occur in vitro, have now been shown to be exhibited in vivo, Mb acting as an NO scavenger under normal physiological conditions, and both Mb and Hb as peroxidases under pathophysiological conditions. Within the last few years, and with the advent of the ability to search genomes, further haemoglobins have been discovered, greatly expanding the family of haem-containing globins. These new globins do not appear to function as oxygen carrier or storage proteins; while some have been assigned to removal of NO, the physiological functions of others remain unclear.
In animals, two new members of this globin family were reported in 2000 and 2002 (Burmester et al. 2000, 2002). These are, respectively, neuroglobin, ubiquitous to all mammalian nervous tissues, and cytoglobin, present throughout the vertebrates. In plants, it has recently been discovered that there are three classes of plant-encoded globins, termed non-symbiotic globins, in addition (in the leguminosae at least) to the symbiotic bacterial leghaemoglobins. Extensive spectroscopic and structural studies on animal Mb and Hb and leghaemoglobin have shown that the haem iron in these proteins is five co-ordinate. The sixth co-ordination site is free to bind ligands such as O2, CO and NO (Fig. 1A). What makes the newly discovered novel plant and animal globins distinct from these is that the haem iron is hexaco-ordinate, the sixth co-ordination site being occupied by a distal histidine (Fig. 1B and C). Despite the presence of a distal ligand, all the proteins are capable of binding oxygen reversibly. Most of these hexaco-ordinate globins also appear to have affinities for oxygen in the nanomolar range or are rapidly autoxidized on contact with oxygen. Thus, these globins do not appear to function as oxygen transport or storage proteins.
Both the animal and the plant hexaco-ordinate globins are upregulated during hypoxia or following ischaemic reperfusion (Trevaskis et al. 1997). Therefore, it has been suggested that there might be a common physiological function for hexaco-ordinate haemoglobin in both plants and animals. The phylogenetically ancient neuroglobins (Ngb) in the brain of humans and mice appear to protect neurones from hypoxia and ischaema–reperfusion damage (Sun et al. 2001; Wakasugi et al. 2005). They may enhance oxygen supply to neurones (Burmester et al. 2002) but have also been postulated to be involved in signal transduction via interaction with G-protein-coupled receptors, O2 sensing, enzymatic activity similar to NADH reductases, or NO detoxification (Dewilde et al. 2001). Cytoglobin (Cgb, also known as histoglobin) is similarly thought to have a role in NO metabolism and detoxification (Hankeln et al. 2005).
Hexaco-ordinate, non-symbiotic, plant haemoglobins are strongly induced in both roots and rosette leaves in hypoxic conditions and may modulate NO bioactivity (Perazzolli et al. 2004). Ferric Arabidopsis Hb1 (AHb1) can be directly but very slowly reduced by NADPH (Perazzolli et al. 2004). On the assumption that there is a relatively efficient reductase system present in vivo, it has been proposed that AHb1 acts as the terminal member of an electron transfer pathway that, on binding oxygen, reacts with NO to form nitrate, oxidizing the haem iron once more (NO dioxygenase activity). It has also been observed that NO accumulates to a high level when AHb1 is silenced. Class-2 non-symbiotic Arabidopsis Hb2 (AHb2) is overexpressed at low temperatures. Seedlings overexpressing AHb2 show enhanced survival in response to hypoxic stress. Combined knockout of both classes of non-symbiotic Hb leads to seedling death. Both proteins are reported to have high oxygen affinity, but while AHb1 forms a stable oxy-complex, AHb2 is rapidly oxidized to the ferric form (Bruno et al. 2007). Both proteins are found within the cytoplasmic compartment, a location consistent with the putative roles suggested at the start of this review, since this compartment is accessible to radicals formed at a variety of sources (e.g. mitochondria, plasma membrane, chloroplast; Mullineaux et al. 2006). The truncated hexaco-ordinated Hb from Mycobacterium tuberculosis is suggested to be a peroxidase and, most excitingly, it is reported that this protein can store two oxidation equivalents, one as a tyrosyl and one as a tryptophanyl radical (Ouellet et al. 2007).
In the past few years, our understanding of haemoglobins has evolved and we now recognize both the NO reductase and peroxidase actions as important functions. The newly discovered haemoglobins in animals and plants seem to function as enzymes, and the depth of their involvement in cellular physiology will surely be further revealed in the next few years.
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