Diversity of Globin Function: Enzymatic, Transport, Storage, and Sensing

Abstract

The availability of genomic information from the three kingdoms of life has altered substantially our view of the globin superfamily. It is now evident that Hbs, 2The abbreviations used are: HbshemoglobinsSDsingle-domainFHbsflavohemoglobinsGCSsglobin-coupled sensors2/2Hbs2/2 α-helical fold HbsSDFgbsSD FHb-like globinsSDSgbsSD sensor globinsMbsmyoglobinsNgbsneuroglobinsCygbscytoglobinsRBCsred blood cellsSNOHbS-nitrosylated HbMetHbmethemoglobinLegHbslegume HbsHBLhexagonal bilayerHemATsaerotactic heme sensorsMCPmethyl-accepting chemotaxis protein. 2The abbreviations used are: HbshemoglobinsSDsingle-domainFHbsflavohemoglobinsGCSsglobin-coupled sensors2/2Hbs2/2 α-helical fold HbsSDFgbsSD FHb-like globinsSDSgbsSD sensor globinsMbsmyoglobinsNgbsneuroglobinsCygbscytoglobinsRBCsred blood cellsSNOHbS-nitrosylated HbMetHbmethemoglobinLegHbslegume HbsHBLhexagonal bilayerHemATsaerotactic heme sensorsMCPmethyl-accepting chemotaxis protein. defined as hemeproteins comprising five to eight α-helices (A–H), with an invariant His at position F8 providing the proximal ligand to the heme iron, occur as three families in two structural classes (1Vinogradov S. Hoogewijs D. Bailly X. Arredondo-Peter R. Gough J. Guertin M. Dewilde S. Moens L. Vanfleteren J. BMC Evol. Biol. 2006; 6: 31-67Crossref PubMed Scopus (169) Google Scholar). Within each family, the Hb can be either chimeric or SD. Historically, the first members of the two families that display the canonical 3/3 α-helical fold were chimeric: the FHbs in Escherichia coli and yeast discovered in ∼1990, consisting of an N-terminal Hb coupled to a ferredoxin reductase-like domain, and the GCSs reported in bacteria and Archaea a decade later, comprising an N-terminal Hb linked to variable gene regulatory domains. The third family of Hbs discovered concomitantly in algae, ciliates, and bacteria were the 2/2Hbs (“truncated” Hbs), which exhibit a 2/2 α-helical fold (see supplemental figure). More recently, SD globins have been discovered in the FHb-like and sensor Hb families that we have called SDFgbs and SDSgbs, respectively (2Vinogradov S. Hoogewijs D. Bailly X. Arredondo-Peter R. Gough J. Dewilde S. Moens L. Vanfleteren J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11385-11389Crossref PubMed Scopus (129) Google Scholar, 3Vinogradov S. Hoogewijs D. Bailly X. Dewilde S. Moens L. Vanfleteren J. Gene (Amst.). 2007; 398: 132-142Crossref PubMed Scopus (90) Google Scholar). Fig. 1 shows diagrammatically the three Hb families and summarizes their distribution in bacteria and eukaryotes. A classification of Hbs is presented in the supplemental table. Only bacteria have representatives of all three families in chimeric and SD guise; the Archaea and eukaryotes lack FHbs and GCSs, respectively. On the basis of the higher sequence similarity to bacterial FHbs/SDFgbs than to GCSs and 2/2Hbs and the presence of FHbs/SDFgbs in unicellular eukaryotes, we have proposed that all eukaryotic Hbs, including vertebrate α/β-globins, Mbs, Ngbs, and Cygbs and all the invertebrate and plant Hbs, emerged from one or more ancestral bacterial SDFgbs (2Vinogradov S. Hoogewijs D. Bailly X. Arredondo-Peter R. Gough J. Dewilde S. Moens L. Vanfleteren J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11385-11389Crossref PubMed Scopus (129) Google Scholar). hemoglobins single-domain flavohemoglobins globin-coupled sensors 2/2 α-helical fold Hbs SD FHb-like globins SD sensor globins myoglobins neuroglobins cytoglobins red blood cells S-nitrosylated Hb methemoglobin legume Hbs hexagonal bilayer aerotactic heme sensors methyl-accepting chemotaxis protein. hemoglobins single-domain flavohemoglobins globin-coupled sensors 2/2 α-helical fold Hbs SD FHb-like globins SD sensor globins myoglobins neuroglobins cytoglobins red blood cells S-nitrosylated Hb methemoglobin legume Hbs hexagonal bilayer aerotactic heme sensors methyl-accepting chemotaxis protein. The variety of Hbs in bacteria makes it clear that the familiar O2 transport function of vertebrate Hbs is a relatively recent adaptation and that the early Hb functions must have been enzymatic and O2-sensing. In this review, we will not discuss O2 transport by animal (metazoan) Hbs; instead, we will focus on the reactions and functions of the FHbs/SDFgbs in the first five sections. The functions of the remaining two globin families will be discussed in the last two sections. The reactions with NO are common to all hemeproteins and likely represent one of the earliest and most important Hb functions. The most significant advances in our understanding of the reactions with NO, shown diagrammatically in Fig. 2, have come in two areas: vertebrate Hbs and Mbs on one hand and bacterial and fungal FHbs on the other. Physiological evidence for the importance of NO reactions with vertebrate Hbs and Mbs is provided by the alterations in the cardiovascular systems of Hb-deficient Antarctic icefish and Mb-deficient mice that are symptomatic of chronic exposure to NO: increased blood flow, hematocrit, tissue vascularization, and mitochondrial density (4di Prisco G. Eastman J. Giordano D. Parisi E. Verde C. Gene (Amst.). 2007; 398: 143-155Crossref PubMed Scopus (41) Google Scholar, 5Kanatous S. Garry D. Respir. Physiol. Neurobiol. 2006; 151: 151-156Crossref PubMed Scopus (13) Google Scholar). In vivo 31P NMR studies of Mb-deficient mouse hearts demonstrated an impaired function of the respiratory chain (6Flögel U. Jacoby C. Godecke A. Schrader J. Magn. Reson. Med. 2007; 57: 50-58Crossref PubMed Scopus (34) Google Scholar), implying NO scavenging by Mb (reactions 3 and 4) (Fig. 2). Furthermore, deoxy-Mb was shown recently to have a nitrite reductase activity (reaction 7) (7Rassaf T. Flögel U. Drexhage C. Hendgen-Cotta U. Kelm M. Schrader J. Circ. Res. 2007; 100: 1749-1754Crossref PubMed Scopus (239) Google Scholar). An ability to shift from NO scavenging at normal oxygen concentrations to generation of NO under hypoxia may allow Mb to act as an O2 sensor to modify the activity of the terminal oxidases and thus the energy status of cardiac cells in response to limited oxygen concentration. Earlier, it was shown that in RBCs, NO is essentially scavenged irreversibly via reactions 3, 4, and 6 (Fig. 2), generating unreactive NO3-. A role of RBCs in hypoxic vasodilation has been proposed in which S-nitrosylation of the β-chain Cys93 residues is favored by the oxy-Hb conformation to form SNOHb, followed by NO transport as SNOHb to peripheral tissues, where release of NO is promoted by the deoxy-Hb conformation (8Singel D. Stamler J. Annu. Rev. Physiol. 2005; 67: 99-145Crossref PubMed Scopus (392) Google Scholar). This proposal remains controversial; in particular, the mechanisms of the NO transfer from heme to Cys and of its release from SNOHb remain elusive. The finding that SNOHb is formed during the reaction of deoxy-Hb with NO2- has led to the suggestion that S-nitrosylation of Cys93 at low oxygen concentrations is carried out by an intermediate in this reaction (9Nagababu E. Ramasamy S. Rifkind J. Nitric Oxide. 2006; 15: 20-29Crossref PubMed Scopus (72) Google Scholar). Consistent with this idea, deoxy-Hb can act as an S-nitrosylated synthase under physiologically relevant conditions (10Angelo M. Singel D. Stamler J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8366-8371Crossref PubMed Scopus (185) Google Scholar). An alternative view, the “Nitrite hypothesis,” proposed that NO2- is an available storage form of NO in RBCs and that Hb generates NO under hypoxic conditions via an enzymatic reduction of NO2- (reaction 7) (11Rifkind J. Nagababu E. Ramasamy S. Antioxid. Redox Signal. 2006; 8: 1193-1203Crossref PubMed Scopus (25) Google Scholar). Very recently, it was proposed that in addition to reaction 7, NO2- can bind to MetHb to form HbFe(III)NO2- and that the latter can react with NO to generate N2O3, with Hb acting as a catalyst (12Basu S. Grubina R. Huang J. Conradie J. Huang Z. Jeffers A. Jiang A. He X. Azarov I. Seibert R. Mehta A. Patel R. King S. Hogg N. Ghosh A. Gladwin M. Kim-Shapiro D. Nat. Chem. Biol. 2007; 3: 785-794Crossref PubMed Scopus (194) Google Scholar): 2NO2-+HbFe(II)+H+→HbFe(II)+N2O3+OH-.N2O3 can nitrosylate Cys, diffuse out of RBCs, and homolyze to NO and NO2*, providing NO for export (12Basu S. Grubina R. Huang J. Conradie J. Huang Z. Jeffers A. Jiang A. He X. Azarov I. Seibert R. Mehta A. Patel R. King S. Hogg N. Ghosh A. Gladwin M. Kim-Shapiro D. Nat. Chem. Biol. 2007; 3: 785-794Crossref PubMed Scopus (194) Google Scholar). The most important, well documented function of FHbs in pathogenic yeast and bacteria is protection against NO toxicity: null mutants exhibit decreased pathogenicity and increased sensitivity to NO. The response to NO exposure is generally complex: in Candida albicans, nine genes are overexpressed, including only one of four FHb genes (13Hromatka B. Noble S. Johnson A. Mol. Biol. Cell. 2005; 16: 4814-4826Crossref PubMed Scopus (145) Google Scholar). E. coli FHb has been studied intensively (14Stevanin T. Read R. Poole R. Gene (Amst.). 2007; 398: 62-68Crossref PubMed Scopus (54) Google Scholar, 15Gardner P. Gardner A. Brashear W. Suzuki T. Hvitved A. Setchell K. Olson J. J. Inorg. Biochem. 2006; 100: 542-550Crossref PubMed Scopus (106) Google Scholar, 16Gardner P. J. Inorg. Biochem. 2005; 99: 247-266Crossref PubMed Scopus (219) Google Scholar); under aerobic conditions, it dioxygenates NO with high efficiency and fidelity (reaction 3) via an enzymatic mechanism (EC 1.14.12.17). The overall reaction is as follows: 2NO+2O2+NAD(P)H↔2NO3-+NAD(P)+H+. The NO dioxygenase activity of FHbs is >20-fold higher than that of either Hb or Mb (16Gardner P. J. Inorg. Biochem. 2005; 99: 247-266Crossref PubMed Scopus (219) Google Scholar). Under anaerobic conditions, FHb exhibits a very much lower NO reductase activity (reaction 5), which may be a significant route for NO decomposition in the absence of other catalysts. In either case, the C-terminal reductase regenerates FHbFe(II) (reaction 8). Although FHb protects pathogenic microorganisms from the host immune systems, it also protects nonpathogenic ones, e.g. the soil firmicute Bacillus subtilis, from NO generated during microaerobic nitrate and nitrite reduction (17Rogstam A. Larsson J. Kjelgaard P. von Wachenfeldt C. J. Bacteriol. 2007; 189: 3063-3071Crossref PubMed Scopus (56) Google Scholar). An additional possibility is that FHb plays a role in the coordination of intracellular NO concentration with extracellular O2 levels, as suggested by the mitochondrial generation of NO in the yeast Saccharomyces exposed to hypoxia, with concomitant localization of the FHb from the cytoplasm to the promitochondrial matrix (18Castello P. David P. McClure T. Crook Z. Poyton R. Cell Metab. 2006; 3: 277-287Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Little is known about reactions of invertebrate Hbs with NO, except for extracellular Hb of nematode Ascaris, the O2 affinity of which is >100-fold higher than that of Mb. Ascaris is adapted to anaerobic life in intestine of the host (PO2 < 10 torr), and the NO dioxygenase activity of its Hb is much smaller than that of FHbs (16Gardner P. J. Inorg. Biochem. 2005; 99: 247-266Crossref PubMed Scopus (219) Google Scholar). Thus, the function of Ascaris Hb is thought to be NO-mediated control of O2 concentration, in contrast to FHbs, which use O2 to control NO concentration, and mammalian Hbs, which employ NO to regulate RBC oxygen delivery (19Gow A. Payson A. Bonaventura J. J. Inorg. Biochem. 2005; 99: 903-911Crossref PubMed Scopus (14) Google Scholar). Mb occurs in cytoplasm of cardiac and skeletal myofibers, and its expression is increased by over an order of magnitude in response to exercise and adaptation to hypoxia, e.g. life at high altitude and in diving mammals (5Kanatous S. Garry D. Respir. Physiol. Neurobiol. 2006; 151: 151-156Crossref PubMed Scopus (13) Google Scholar). Its role was thought to be O2 delivery via facilitated diffusion to terminal oxidases (20Wittenberg J. Gene (Amst.). 2007; 398: 156-161Crossref PubMed Scopus (18) Google Scholar). 1H NMR studies of perfused rat myocardium show only oxy-Mb in resting muscle, which at onset of muscle contraction desaturates to a steady-state level within ∼30 s, indicating a transient role of O2 supply. Equipoise PO2, defined as PO2 at which free and Mb-facilitated diffusions contribute equally to the O2 flux, was calculated to be 1.7 torr at 35 °C and 0.19 mm Mb from the diffusion coefficient and P50 ∼ 2 torr (21Lin P. Kreutzer U. Jue T. Biophys. J. 2007; 92: 2608-2620Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Because PO2 in myocardium in vivo is >10 torr, the Mb contribution is not appreciable; however, in diving mammals, with 10-fold higher Mb concentration, it should be very significant. Hypoxia-dependent expression of carp Mb in nonmuscle tissues hints at additional Mb functions (22Fraser J. de Mello L. Ward D. Rees H. Williams D. Fang Y. Brass A. Gracey A. Cossins A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2977-2981Crossref PubMed Scopus (129) Google Scholar). LegHbs are symbiotic Hbs induced during nodule development resulting from infection by nitrogen-fixing Rhizobium sp. (α-Proteobacteria) or Frankia sp. (Actinomycetes) (23Hebelstrup K.H. Igamberdiev A.U. Hill R.D. Gene (Amst.). 2007; 398: 86-93Crossref PubMed Scopus (72) Google Scholar). Their function is to maintain a low enough free O2 concentration to avoid inactivation of the O2-labile nitrogenase enzyme and to provide sufficient O2 to the terminal oxidases. Comparison with Mb suggests that facilitated diffusion is unlikely to be significant because the ∼10-fold lower P50 offsets the ∼3-fold higher symbiotic Hb concentration. The higher O2 affinity of LegHb (see supplemental material) is matched by the increased O2 affinity of the terminal oxidase. Unlike in the Mb-deficient mouse, where the effect of Mb absence is abrogated by several compensatory mechanisms, RNA interference inhibition of LegHb formation abolishes N2 fixation (24Ott T. van Dongen J. Gunther C. Krusell L. Desbrosses G. Vigeolas H. Bock V. Czechowski T. Geigenberger P. Udvardi M. Curr. Biol. 2005; 29: 531-535Abstract Full Text Full Text PDF Scopus (260) Google Scholar). The SD FHb-like Hb of the β-proteobacterium Vitreoscilla, an obligate aerobe that inhabits O2-depleted environments, exhibits 30–50% identity to bacterial FHbs and is expressed in response to hypoxia (25Chi P. Webster D. Stark B. Microbiol. Res. 2008; 163 (in press)PubMed Google Scholar). It binds to respiratory membranes, specifically to subunit I of cytochrome bo oxidase, supporting the notion that it provides O2 directly to the terminal oxidase(s) to improve respiration and ATP production. The NO dioxygenase activity of Vitreoscilla SDFgb is ∼100-fold smaller than that of FHb (15Gardner P. Gardner A. Brashear W. Suzuki T. Hvitved A. Setchell K. Olson J. J. Inorg. Biochem. 2006; 100: 542-550Crossref PubMed Scopus (106) Google Scholar). Coupling of Vitreoscilla SDFgb with a flavin reductase domain produces the functional equivalent of an FHb NO dioxygenase. Thus, the presence of separate globin and reductase may allow the Vitreoscilla SDFgb to perform dual functions: provision of O2 to the terminal oxidases and protection against NO. Because interaction with phospholipid membranes results in >20-fold decreases in O2 and NO affinities, lipid binding may be used to modulate the affinities of Hb. Furthermore, there are differences between bacterial FHbs and SDFgbs, including Vitreoscilla SDFgb: although the autoxidation rates and O2 and NO binding affinities of both groups are higher compared with Mb, the FHbs are less stable and have lower affinities than the SDFgbs (26Farres J. Rechsteiner M. Herold S. Frey A. Kallio P. Biochemistry. 2005; 44: 4125-4134Crossref PubMed Scopus (37) Google Scholar). Numerous studies have found that heterologous expression of Vitreoscilla SDFgb substantially enhances growth and protein synthesis, particularly under O2-limited conditions. Consequently, it has become an important tool in biotechnology used to improve production of biologicals, e.g. α-amylase in B. subtilis and erythromycin in Saccharopolyspora erythraea, and to increase degradation of benzoic acid derivatives by Burkholderia sp. (27Urgun-Demirtas M. Stark B. Pagilla K. Crit. Rev. Biotechnol. 2006; 26: 145-164Crossref PubMed Scopus (89) Google Scholar). Metazoan Hbs and Mbs have peroxidase activity and can catalyze the oxidation of biological molecules using H2O2 (28Reeder B. Wilson M. Curr. Med. Chem. 2005; 12: 2741-2751Crossref PubMed Scopus (122) Google Scholar). The first step is the formation of the met (ferric) form either by autoxidation (reaction 1) (Fig. 3) or by reaction with H2O2 via reactions 2 and 3. Next, the met form reacts with peroxides to produce a ferryl radical, HP+·Fe(IV)OH- (with HP representing hemeprotein), where the free radical resides on the porphyrin ring or specific Tyr/Trp residues and is unstable, unlike in classical peroxidases. The ferryl iron and the radical are both active and may oxidize appropriate substrates, e.g. membrane lipids, as well as form heme-to-protein cross-linked derivatives (reaction 4), via the distal His (29Reeder B. Cutruzzolà F. Bigotti M. Watmough N. Wilson M. IUBMB Life. 2007; 59: 477-489Crossref PubMed Scopus (17) Google Scholar), which are also reactive. The oxidation of lipids produces active radicals, e.g. peroxyl (LOO•) and hydroperoxides (LOOH), which can react with MetHb to form additional ferryl species. Hence, the Hb and Mb peroxidase activities initiate a cascade of lipid oxidations, particularly with polyunsaturated fatty acids, generating prostaglandin-like molecules with potent vasoconstrictor activity (28Reeder B. Wilson M. Curr. Med. Chem. 2005; 12: 2741-2751Crossref PubMed Scopus (122) Google Scholar). E. coli FHb is inactive toward peroxidase substrates, and the ferric FHb does not react with H2O2. However, the latter binds unsaturated or cyclopropanated fatty acids and acts as an alkylhydroperoxide reductase with NADH as electron donor, catalyzing the reduction of alkylhydroperoxides to the corresponding alcohols (30D'Angelo P. Lucarelli D. della Longa S. Benfatto M. Hazemann J.L. Feis A. Smulevich G. Ilari A. Bonamore A. Boffi A. Biophys. J. 2004; 86: 3882-3892Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). These results suggest that FHb may be involved in the repair of oxidatively damaged lipid membranes. Although Ralstonia eutropha FHb also binds lipids, the yeast Saccharomyces cerevisiae FHb does not, implying that lipid binding may occur with bacterial but not fungal FHbs. Autoxidation of oxy-Hb forms O2-, which can react with NO to generate peroxynitrite (ONOO–), which unlike O2-, can move across cellular membranes. Although formation of ONOO– is unlikely to be significant in RBCs, it may be important in the case of cytoplasmic Hbs, e.g. vertebrate Ngbs, which have high autoxidation rates; NgbFe(II)NO reacts with ONOO– ∼100-fold faster compared with Hb, followed by dissociation to NO and MetNgb (31Herold S. Fago A. Comp. Biochem. Physiol. A. 2005; 142: 124-129Crossref PubMed Scopus (77) Google Scholar). Such reactions may have a protective function in cerebral ischemia. Pathogenic bacteria have to contend not only with NO, but also with O2- generated by macrophages and hence with ONOO–. The latter can react with CO2 to produce additional highly reactive free radicals, e.g. trioxocarbonate (a•CO3-) and nitrogen dioxide (•NO2). Thus, bacterial Hbs may play a role in the detoxification of free radicals other than NO (32Wu G. Wainwright L. Poole R. Adv. Microb. Physiol. 2003; 47: 255-310Crossref PubMed Scopus (104) Google Scholar). The polychaete Amphitrite inhabits coastal mud flats, where it is exposed to toxic halophenols secreted by other worms. In addition to an extracellular HBL Hb, it has two intracellular Hbs, which act as peroxidases to oxidatively dehalogenate the halophenols to the corresponding, much less toxic quinones by a novel mechanism (33Franzen S. Gilvey L.B. Belyea J. Biochim. Biophys. Acta. 2007; 1774: 121-130Crossref PubMed Scopus (51) Google Scholar). Ngbs and Cygbs occur widely in vertebrates and represent the two lineages of vertebrate globins, with the α- and β-globins and Mbs being part of the Cygb lineage. Ngb is found in the cytoplasm of neuronal and endocrine tissues, and Cygb in retinal neurons and fibroblast-like cells in internal organs (34Brunori M. Vallone B. CMLS Cell. Mol. Life Sci. 2007; 64: 1259-1268Crossref PubMed Scopus (90) Google Scholar). Their concentrations are in the μm range, except for Ngb in retina, where it can be >10-fold higher. In contrast to the majority of vertebrate globins, which are pentacoordinated, Ngbs and Cygbs are hexacoordinated, with the distal His(E7) coordinating directly with the heme iron (35Halder P. Trent J. Hargrove M. Proteins. 2007; 66: 172-182Crossref PubMed Scopus (44) Google Scholar). The binding of exogenous ligands requires movement of the distal His(E7) out of the way, thus providing a conformational alteration that could assist in the modulation of enzymatic activity and that could be useful in sensing. There is general agreement that Ngb plays a neuroprotective role during hypoxic stress (34Brunori M. Vallone B. CMLS Cell. Mol. Life Sci. 2007; 64: 1259-1268Crossref PubMed Scopus (90) Google Scholar, 36Fordel E. Thijs L. Moens L. Dewilde S. FEBS J. 2007; 274: 1312-1317Crossref PubMed Scopus (114) Google Scholar). It lacks O2 and NO reductase activities, but shows NO dioxygenase activity (reaction 3) (Fig. 2) and may function as a sensor of the relative O2 and NO concentrations. Its up-regulation in hypoxia, followed by a decrease upon reoxygenation, implies scavenging of reactive oxygen species (36Fordel E. Thijs L. Moens L. Dewilde S. FEBS J. 2007; 274: 1312-1317Crossref PubMed Scopus (114) Google Scholar). Cygb is induced in fibrosis and may regulate collagen gene expression. Exposure to hypoxia increases the expression of Cygb in the retina, suggesting a role in retinal O2 homeostasis. Furthermore, exposure of neuroblastoma cells to H2O2, but not other stimuli, elicits up-regulation of Cygb, and transfection with Cygb small interfering RNA vectors promotes cell death (37Li D. Chen X. Li W. Yang Y. Wang J. Yu A. Neurochem. Res. 2007; 32: 1375-1380Crossref PubMed Scopus (83) Google Scholar). In amphibians, which lack Mb, Cygb appears to be expressed in heart and skeletal muscles (38Xi Y. Obara M. Ishida Y. Ikeda S. Yoshizato K. Gene (Amst.). 2007; 398: 94-102Crossref PubMed Scopus (25) Google Scholar). Plant Hbs are subdivided into two classes, both comprising symbiotic Hbs and nonsymbiotic Hbs, with the symbiotic LegHbs being part of class II. The nonsymbiotic Hbs are hexacoordinated, occur in μm concentrations in various plant tissues, and have O2 binding affinities >10-fold higher compared with Mb (see supplemental material). Although overexpression of class I nonsymbiotic Hb genes in hypoxia is a general property, the effect is indirect and involves interaction with NO (23Hebelstrup K.H. Igamberdiev A.U. Hill R.D. Gene (Amst.). 2007; 398: 86-93Crossref PubMed Scopus (72) Google Scholar). In addition, they also play a role in the dioxygenation of NO, a reaction shown to be important in maintaining the cellular redox and energy status via the Hb/NO cycle, with the Hb contributing to NAD(P)H oxidation by turnover of NO (23Hebelstrup K.H. Igamberdiev A.U. Hill R.D. Gene (Amst.). 2007; 398: 86-93Crossref PubMed Scopus (72) Google Scholar). Because NO is now recognized to be an important plant hormone, the nonsymbiotic Hbs may function in the regulation of NO metabolism and cell signaling. Arabidopsis class II nonsymbiotic Hbs appear to scavenge NO and play a role in seedling development (39Hebelstrup K. Hunt P. Dennis E. Jensen S. Jensen E. Physiol. Plant. 2006; 127: 157-166Crossref Scopus (61) Google Scholar). In H2S-rich environments, at sea level and in hydrothermal vents on the ocean floor, bivalves and annelids form thioautotrophic symbioses with sulfur-oxidizing γ-Proteobacteria located in specialized gill cells in the former case or in a special organ, the trophosome, in the latter (40Weber R. Vinogradov S. Physiol. Rev. 2001; 181: 568-629Google Scholar). The digestive system of the host is either absent or highly reduced, and the endosymbiont oxidizes H2S and fixes carbon to provide for all the metabolic requirements of the host. The clams Lucina and Solemya have cytoplasmic Hbs that divide the function of transporting O2 and H2S to the symbiont. Lucina pectinata HbI binds H2Sto form ferric Hb sulfide with an affinity orders of magnitude higher compared with Mb and the other two Hbs. The hydrothermal vent annelid Riftia has three extracellular Hbs, two in the vascular (3.5-MDa HBL and 400-kDa Hbs) and a 400-kDa Hb in the coelomic compartments, with all three sharing most of the constituent globin chains (40Weber R. Vinogradov S. Physiol. Rev. 2001; 181: 568-629Google Scholar). In the vascular system, where [H2S] ∼ 2–12 mm, the HBL Hb is thought to transport H2S bound to its Cys residues (41Bailly X. Vinogradov S. J. Inorg. Biochem. 2005; 99: 142-150Crossref PubMed Scopus (35) Google Scholar). Although this view was challenged by the proposal that sulfide binds to interior zinc ions in the coelomic Hb, the absence of zinc in the crystal structure of the 400-kDa Hb from the pogonophoran Oligobrachia inhabiting similar environments (42Numoto N. Nakagawa T. Kita A. Sasayama Y. Fukumori Y. Miki K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14521-14526Crossref PubMed Scopus (35) Google Scholar) implies that there may be more than one way to transport H2S. Members of the GCS family, including SDSgbs and distantly related protoglobins, occur in ∼30% of bacterial globin-containing genomes. The GCSs can be subdivided into HemATs with MCP-related C-terminal domains and gene regulators with undefined C-terminal domain function(s) (43Freitas T. Saito J. Hou S. Alam M. J. Inorg. Biochem. 2005; 99: 23-33Crossref PubMed Scopus (76) Google Scholar). Although MCPs are chimeric transmembrane proteins with N-terminal periplasmic sensing and cytoplasmic signaling domains, the HemATs are soluble proteins with C-terminal domains exhibiting ∼30% identity to other MCP domains. The HemATs can elicit both positive and negative aerotactic responses: aerophilic in the case of B. subtilis and aerophobic in the Archaea Halobacterium salinarum (44Zhang W. Olson J. Phillips G. Biophys. J. 2005; 88: 2801-2814Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The B. subtilis wild-type HemAT and its sensor domain both exhibit biphasic O2 binding, with low and high affinity components (48Ouellet H. Ranguelova K. Labarre M. Wittenberg J. Wittenberg B. Magliozzo R. Guertin M. J. Biol. Chem. 2007; 282: 7491-7503Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Thus, HemAT can respond over a broad range of O2 concentrations, from hypoxic (0–10 μm) to aerobic (>50 μm). Nothing is known about the function of the bacterial SDSgbs; to function as sensors, they would have to form complexes with MCP-like or other signaling proteins. The protoglobins have an Cys(E19) similar to the H2S-binding annelid HBL Hbs; thus, their function may be other than sensing. 2/2Hbs (truncated Hbs) occur in ∼71% of bacterial globin-containing genomes in three classes; 2/2Hb1s occur also in Archaea, green algae, ciliates, and diatoms, and 2/2Hb2s are found in plants and diatoms (see supplemental material) (1Vinogradov S. Hoogewijs D. Bailly X. Arredondo-Peter R. Gough J. Guertin M. Dewilde S. Moens L. Vanfleteren J. BMC Evol. Biol. 2006; 6: 31-67Crossref PubMed Scopus (169) Google Scholar). The crystal structures are characterized by an absent or vestigial A helix and replacement of the F helix by a loop with a single-turn helix (45Nardini M. Pesce A. Milani M. Bolognesi M. Gene (Amst.). 2007; 398: 2-11Crossref PubMed Scopus (61) Google Scholar). The 2/2Hb1s and 2/2Hb2s, but not the 2/2Hb3s, have a protein matrix tunnel/cavity vicinal to the heme, which is thought to affect ligand movement to and from the heme and thus play a role in their ligand binding. Mycobacterium sp. (Actinobacteria) contain one to three genes, glbN, glbO, and glbP, which encode 2/2Hb1–3s, respectively, as well as FHbs (46Ascenzi P. Bolognesi M. Milani M. Guertin M. Visca P. Gene (Amst.). 2007; 398: 42-51Crossref PubMed Scopus (46) Google Scholar). Although Mycobacterium tuberculosis 2/2Hb1 and 2/2Hb2 have ∼100-fold higher O2 affinities compared with Mb, they differ widely in their NO dioxygenation activities: the former is comparable with E. coli FHb, and the latter is ∼50-fold smaller than Mb (14Stevanin T. Read R. Poole R. Gene (Amst.). 2007; 398: 62-68Crossref PubMed Scopus (54) Google Scholar). 2/2Hb1 is thought to scavenge NO because heterologous expression in E. coli increases NO consumption, and knocking out the glbN gene causes a dramatic reduction in the latter. Heterologous expression of M. tuberculosis 2/2Hb1 and 2/2Hb2 in FHb-deficient mutants of Salmonella showed that the former, but not the latter, increased the survival of the host cells within rat peritoneal macrophages (47Pawaria S. Rajamohan G. Gambhir V. Lama A. Varshney G. Dikshit K. Microb. Pathog. 2007; 42: 119-128Crossref PubMed Scopus (29) Google Scholar). 2/2Hb2 may have an oxidation/reduction function because it has peroxidase activity with formation of ferryl intermediates (48Ouellet H. Ranguelova K. Labarre M. Wittenberg J. Wittenberg B. Magliozzo R. Guertin M. J. Biol. Chem. 2007; 282: 7491-7503Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Mycobacterium leprae is an interesting case because it is known to have undergone reductive evolution: it only has 2/2Hb2, which is capable of protecting it against NO as well as oxidative stress. The Actinobacteria Streptomyces and Frankia have chimeric 2/2Hb2s with an N-terminal ∼100-amino acid monooxygenase domain; additional Hbs are present in both (49Bonamore A. Attili A. Arenghi F. Catacchio B. Chiancone E. Morea V. Boffi A. Gene (Amst.). 2007; 398: 52-61Crossref PubMed Scopus (14) Google Scholar). Streptomyces avermitilis oxy-2/2Hb2 can oxidize small quinols and may protect against environmental quinones. The microaerophilic ϵ-proteobacterium Campylobacter jejuni, the causative agent of gastroenteritis, has a 2/2Hb3 and an SD globin. The NO dioxygenase activity of the latter is comparable with that of Vitreoscilla Hb (15Gardner P. Gardner A. Brashear W. Suzuki T. Hvitved A. Setchell K. Olson J. J. Inorg. Biochem. 2006; 100: 542-550Crossref PubMed Scopus (106) Google Scholar). Although neither are essential for growth, knock-out 2/2Hb3 mutants have decreased respiration rates, and SDFgb mutants exhibit hypersensitivity to NO (50Lu C. Egawa T. Wainwright L. Poole R. Yeh S. J. Biol. Chem. 2007; 282: 13627-13636Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). At present, we have no clue about the evolutionary relationships between the three globin families. It is likely that the earliest functions of globins involved O2 scavenging and sensing and reactions with NO. In extant unicellular prokaryotes and eukaryotes, members of the FHb/SDFgb and 2/2Hb families function as enzymes, whereas in Archaea and bacteria, the GCSs function as aerotactic O2 sensors and gene regulators. The emergence of multicellular animals and plants was accompanied by extensive diversification of the enzymatic functions of SD globin, including adaptations to reversible O2 binding, permitting its transport and storage. There is agreement that in vertebrates, the Ngbs and Cygbs evolved from a common ancestral SDFgb and that Mbs and the α- and β-globins split off from the Cygb branch (34Brunori M. Vallone B. CMLS Cell. Mol. Life Sci. 2007; 64: 1259-1268Crossref PubMed Scopus (90) Google Scholar). Likewise, plant Hbs emerged from an ancestral SDFgb, evolving into nonsymbiotic Hbs with enzymatic functions and LegHbs, symbiotic Hbs able to bind O2 reversibly. Whether the metazoan and plant Hbs emerged from an ancestral eukaryotic SDFgb and the relationship of the latter to the bacterial SDFgbs remain to be elucidated. The 2/2Hbs appear to have preserved their enzymatic function(s) in the prokaryotes, plants, and a few unicellular eukaryotes without evolving any nonenzymatic function. Likewise, the sensor globin family appears to have maintained its O2-sensing function without adaptations to other functions. Thus, the FHb/SDFgb family was able to diversify and adapt its functions much more extensively than the other two families, ensuring its extraordinary evolutionary success in multicellular organisms. We have proposed a model of globin evolution based on two premises (3Vinogradov S. Hoogewijs D. Bailly X. Dewilde S. Moens L. Vanfleteren J. Gene (Amst.). 2007; 398: 132-142Crossref PubMed Scopus (90) Google Scholar). The first is that all three globin families emerged and evolved in bacteria over the time span from the origin of life (4100 to 3500 megayears ago) to the endosymbiotic acquisition of plastids and mitochondria by unicellular eukaryotes ∼1600 to 1000 megayears ago. The second is that lateral transfer(s) of FHb/SDFgb gene(s) to unicellular eukaryote(s), coincident with or separate from the endosymbiotic events and prior to the emergence of multicellularity (∼900 to 650 megayears ago), provided the ancestor(s) of all the eukaryote globin genes. Within this framework, the early globin functions in bacteria, in mostly anoxic environments, would have been enzymatic scavenging of NO and/or O2 and sensing of O2 concentration gradients. The many additional functions we observe today, exemplified by O2 transport and storage, allosteric O2 binding, H2S binding and transport, and dehaloperoxidase activity, would have evolved subsequent to the emergence of multicellularity concomitant with increases in atmospheric O2 levels within the last ∼650 megayears. We thank Drs. A. Fago, P. Gardner, P. Hunt, R. Johnson, B. Reeder, A. Riggs, and B. Stark for helpful comments. Download .pdf (.56 MB) Help with pdf files