Heme-containing Proteins Research Articles

Structural determinants of ligand binding in truncated hemoglobins: Resonance Raman spectroscopy of the native states and their carbon monoxide and hydroxide complexes

The ligand binding characteristics of heme-containing proteins are determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. In this regard, the heme pockets of truncated hemoglobins (TrHbs) constitute an interesting case study as they share many common features, including a number of polar cavity residues. In this review, we will focus on three proteins of group II TrHbs, from Thermobifida fusca (Tf-HbO) and Pseudoalteromonas haloplanktis TAC125 (Ph-HbO). Although the residues in positions G8 (Trp) and B10 (Tyr) are conserved in all three proteins, the CD1 residue is a Tyr in T. fusca and a His in P. haloplanktis. Comparison of the ligand binding characteristics of these proteins, in particular the hydroxo and CO ligands by means of resonance Raman spectroscopy, reveals that this single difference in the key heme cavity residues markedly affects their ligand binding capability and conformation. Furthermore, although the two Ph-HbOs (Ph-HbO-2217 and Ph-HbO-0030) have identical key cavity residues, they display distinct ligand binding properties.

Corynebacterium diphtheriae Iron-Regulated Surface Protein HbpA Is Involved in the Utilization of the Hemoglobin-Haptoglobin Complex as an Iron Source.

Corynebacterium diphtheriae utilizes various heme-containing proteins, including hemoglobin (Hb) and the hemoglobin-haptoglobin complex (Hb-Hp), as iron sources during growth in iron-depleted environments. The ability to utilize Hb-Hp as an iron source requires the surface-anchored proteins HtaA and either ChtA or ChtC. The ability to bind hemin, Hb, and Hb-Hp by each of these C. diphtheriae proteins requires the previously characterized conserved region (CR) domain. In this study, we identified an Hb-Hp binding protein, HbpA (38.5 kDa), which is involved in the acquisition of hemin iron from Hb-Hp. HbpA was initially identified from total cell lysates as an iron-regulated protein that binds to both Hb and Hb-Hp in situ HbpA does not contain a CR domain and has sequence similarity only to homologous proteins present in a limited number of C. diphtheriae strains. Transcription of hbpA is regulated in an iron-dependent manner that is mediated by DtxR, a global iron-dependent regulator. Deletion of hbpA from C. diphtheriae results in a reduced ability to utilize Hb-Hp as an iron source but has little or no effect on the ability to use Hb or hemin as an iron source. Cell fractionation studies showed that HbpA is both secreted into the culture supernatant and associated with the membrane, where its exposure on the bacterial surface allows HbpA to bind Hb and Hb-Hp. The identification and analysis of HbpA enhance our understanding of iron uptake in C. diphtheriae and indicate that the acquisition of hemin iron from Hb-Hp may involve a complex mechanism that requires multiple surface proteins.IMPORTANCE The ability to utilize host iron sources, such as heme and heme-containing proteins, is essential for many bacterial pathogens to cause disease. In this study, we have identified a novel factor (HbpA) that is crucial for the use of hemin iron from the hemoglobin-haptoglobin complex (Hb-Hp). Hb-Hp is considered one of the primary sources of iron for certain bacterial pathogens. HbpA has no similarity to the previously identified Hb-Hp binding proteins, HtaA and ChtA/C, and is found only in a limited group of C. diphtheriae strains. Understanding the function of HbpA may significantly increase our knowledge of how this important human pathogen can acquire host iron that allows it to survive and cause disease in the human respiratory tract.

Characterization of a Novel Porin-Like Protein, ExtI, from Geobacter sulfurreducens and Its Implication in the Reduction of Selenite and Tellurite.

The extI gene in Geobacter sulfurreducens encodes a putative outer membrane channel porin, which resides within a cluster of extHIJKLMNOPQS genes. This cluster is highly conserved across the Geobacteraceae and includes multiple putative c-type cytochromes. In silico analyses of the ExtI sequence, together with Western blot analysis and proteinase protection assays, showed that it is an outer membrane protein. The expression level of ExtI did not respond to changes in osmolality and phosphate starvation. An extI-deficient mutant did not show any significant impact on fumarate or Fe(III) citrate reduction or sensitivity to β-lactam antibiotics, as compared with those of the wild-type strain. However, extI deficiency resulted in a decreased ability to reduce selenite and tellurite. Heme staining analysis revealed that extI deficiency affects certain heme-containing proteins in the outer and inner membranes, which may cause a decrease in the ability to reduce selenite and tellurite. Based on these observations, we discuss possible roles for ExtI in selenite and tellurite reduction in G. sulfurreducens.

Enzymes serve up diverse cyclopropanes

With some engineering in the lab, a quartet of iron-containing heme proteins from microbes can convert inert alkenes into each possible stereoisomer of cyclopropanes, which are valuable motifs in medicinally active compounds (ACS Cent. Sci. 2018, DOI: 10.1021/acscentsci.7b00548). Previous engineered proteins needed help from an artificial cofactor to complete this feat. This work suggests that heme proteins are perfectly capable of doing this chemistry on their own. Building cyclopropanes with protein catalysts is not new, says Frances H. Arnold, the California Institute of Technology professor who led the work. However, prior heme protein catalysts made by her group and others worked best on relatively reactive alkenes. “These proteins are being commercialized, and our clients want more challenging cyclopropanations,” including transformations of unactivated alkenes, she says. So graduate student Anders M. Knight and colleagues used directed evolution, which simulates natural selection, to find promising candidates. They optimized four heme-containing proteins from

Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources.

Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS. This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis ΔiscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.

The Small RNA RyhB Is a Regulator of Cytochrome Expression in Shewanella oneidensis.

Shewanella oneidensis produces an extensive electron transfer network that results in metabolic flexibility. A large number of c-type cytochromes are expressed by S. oneidensis and these function as the fundamental electron transport chain proteins. Although several S. oneidensis cytochromes have been well-characterized, little is known about how their expression is regulated. In this study, we investigate the role of the ferric uptake regulator (Fur) and the sRNA RyhB in regulation. Our results demonstrate that loss of Fur leads to diminished growth and an apparent decrease in heme-containing proteins. Remarkably, deleting the Fur-repressed ryhB gene almost completely reverses these physiological changes, indicating that the phenotypes resulting from loss of Fur are (at least partially) dependent on RyhB. RNA sequencing identified a number of possible RyhB repressed genes. A large fraction of these encode c-type cytochromes, among them two of the most abundant periplasmic cytochromes CctA (also known as STC) and ScyA. We show that RyhB destabilizes the mRNA of four of its target genes, cctA, scyA, omp35, and nrfA and this requires the presence of the RNA chaperone Hfq. Iron limitation decreases the expression of the RyhB target genes cctA and scyA and this regulation relies on the presence of both Fur and RyhB. Overall, this study suggests that controlling cytochrome expression is of importance to maintain iron homeostasis and that sRNAs molecules are important players in the regulation of fundamental processes in S. oneidensis MR-1.

Hemoglobin catalyzes CoA degradation and thiol addition to flavonoids

In the presence of CoA, cell-free extracts prepared from porcine liver was found to convert 7,8-dihydroxyflavone (DHF) to a pantetheine conjugate, which was a novel flavonoid. We purified a 7,8-DHF-converting enzyme from the extracts, and identified it as hemoglobin (Hb). The purified Hb showed the following two activities: (i) degradation of CoA into pantetheine through hydrolytic cleavage to yield pantetheine and 3′-phospho-adenosine-5′-diphosphate (ADP) independently of heme, and (ii) addition of a thiol (e.g., pantetheine, glutathione and cysteine) to 7,8-DHF through C-S bond formation. Human Hb also exhibited the above flavonoid-converting activity. In addition, heme-containing enzymes such as peroxidase and catalase added each of pantetheine, glutathione and cysteine to the flavonoid, although no pantetheine conjugates were synthesized when CoA was used as a substrate. These findings indicated that the thiol-conjugating activity is widely observed in heme-containing proteins. On the other hand, only Hb catalyzed the hydrolysis of CoA, followed by the thiol conjugation to synthesize the pantetheine conjugate. To the best of our knowledge, this is the first report showing that Hb has the catalytic ability to convert naturally occurring bioactive compounds, such as dietary flavonoids, to the corresponding conjugates in the presence of thiol donors or CoA.

Molecular and in silico cloning, identification, and preharvest period expression analysis of a putative cytochrome P450 monooxygenase gene from Camellia sinensis (L.) Kuntze (tea).

Cytochrome P450 monooxygenases are one of the largest heme-containing protein groups, and the majority of them catalyze hydroxylation reactions dependent on nicotinamide adenine dinucleotide phosphate and oxygen. Cytochrome P450 (CYP) enzymes function in a wide range of monooxygenation reactions essential in primary and secondary metabolism in plants. Camellia sinensis (L.) Kuntze is a commercially and economically valuable plant due to its medicinally important secondary metabolites and as a beloved beverage. Cytochrome P450 monooxygenases play a significant role in the biosynthesis of a variety of secondary metabolites in tea. Although the biosynthesis of secondary metabolites has been investigated in detail, there have been limited studies conducted on identifying the genetic mechanisms of CYP-catalyzed secondary metabolic pathways in the C. sinensis (tea) plant. In our study, we characterized a putative C. sinensis (L.) Kuntze cytochrome P450 monooxygenase gene (Csp450), which has 1759 bp full-length cDNA with 49 bp of 5' and 183 bp of 3' untranslated regions. eTh CDS of the gene is 1527 bp and 508 amino acids in length. BLAST results of the deduced amino acid sequence revealed a high similarity with the CYP704C1-like superfamily. Preharvest period gene expression analysis from May, July, and September did not show any difference.

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins.

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron–oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs.

Cyclopropanations via Heme Carbenes: Basic Mechanism and Effects of Carbene Substituent, Protein Axial Ligand, and Porphyrin Substitution.

Catalytic carbene transfer to olefins is a useful approach to synthesize cyclopropanes, which are key structural motifs in many drugs and biologically active natural products. While catalytic methods for olefin cyclopropanation have largely relied on rare transition-metal-based catalysts, recent studies have demonstrated the promise and synthetic value of iron-based heme-containing proteins for promoting these reactions with excellent catalytic activity and selectivity. Despite this progress, the mechanism of iron-porphyrin and hemoprotein-catalyzed olefin cyclopropanation has remained largely unknown. Using a combination of quantum chemical calculations and experimental mechanistic analyses, the present study shows for the first time that the increasingly useful C=C functionalizations mediated by heme carbenes feature an FeII-based, nonradical, concerted nonsynchronous mechanism, with early transition state character. This mechanism differs from the FeIV-based, radical, stepwise mechanism of heme-dependent monooxygenases. Furthermore, the effects of the carbene substituent, metal coordinating axial ligand, and porphyrin substituent on the reactivity of the heme carbenes was systematically investigated, providing a basis for explaining experimental reactivity results and defining strategies for future catalyst development. Our results especially suggest the potential value of electron-deficient porphyrin ligands for increasing the electrophilicity and thus the reactivity of the heme carbene. Metal-free reactions were also studied to reveal temperature and carbene substituent effects on catalytic vs noncatalytic reactions. This study sheds new light into the mechanism of iron-porphyrin and hemoprotein-catalyzed cyclopropanation reactions and it is expected to facilitate future efforts toward sustainable carbene transfer catalysis using these systems.

The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6.

Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α–helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein “halves” for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.

Structural and functional effects of cytochrome b5 interactions with human cytochrome P450 enzymes

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5 Mutations of key b5 residues suggest not only that different b5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

The mitochondrial ABC transporter Atm1 plays a role in iron metabolism and virulence in the human fungal pathogen Cryptococcus neoformans.

Iron-sulfur clusters (ISC) are indispensable cofactors for essential enzymes in various cellular processes. In the model yeast Saccharomyces cerevisiae, the precursor of ISCs is exported from mitochondria via a mitochondrial ABC transporter Atm1 and used for cytosolic and nuclear ISC protein assembly. Although iron homeostasis has been implicated in the virulence of the human fungal pathogen Cryptococcus neoformans, the key components of the ISC biosynthesis pathway need to be fully elucidated. In the current study, a homolog of S. cerevisiae Atm1 was identified in C. neoformans, and its function was characterized. We constructed C. neoformans mutants lacking ATM1 and found that deletion of ATM1 affected mitochondrial functions. Furthermore, we observed diminished activity of the cytosolic ISC-containing protein Leu1 and the heme-containing protein catalase in the atm1 mutant. These results suggested that Atm1 is required for the biosynthesis of ISCs in the cytoplasm as well as heme metabolism in C. neoformans. In addition, the atm1 mutants were avirulent in a murine model of cryptococcosis. Overall, our results demonstrated that Atm1 plays a critical role in iron metabolism and virulence for C. neoformans.

Probing the Role of the Heme Distal and Proximal Environment in Ligand Dynamics in the Signal Transducer Protein HemAT by Time-Resolved Step-Scan FTIR and Resonance Raman Spectroscopy.

HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved step-scan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe-bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full-length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe-His mode of the photoproduct of sensor domain HemAT-CO is detected at a frequency higher than that of the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.

Conjugation with 20 kDa dextran decreases the autoxidation rate of bovine hemoglobin

Haemoglobin-based oxygen carrier (HBOC) is highly susceptible to autoxidation that renders a series of tissue and cellular toxicities. HBOC is prepared by chemical modification of haemoglobin (Hb), which typically increases the autoxidation rate of Hb. Thus, it is necessary to decrease the autoxidation of HBOC. In the present study, two dextran-bHb conjugates (dex20-bHb and dex40-bHb) were prepared by conjugation with 20 kDa or 40 kDa dextrans, where the thiol group of Cys-93(β) was reversibly protected. The autoxidation rate of bHb was decreased by conjugation with 20 kDa dextran and maintained by conjugation with 40 kDa dextran. In order to understand the low autoxidation rate of dex20-bHb, the effects of aldehyde modification and dextran on the autoxidation rate of dex20-bHb were investigated. The high oxygen affinity, high tetramer stability and dextran itself were found to decrease the autoxidation rate of dex20-bHb. The conjugated dextran had a predominant effect on decreasing the autoxidation rate of bHb, which was particularly promising for the potential development of safe HBOCs. Conjugation with dextran is of general significance to decrease the oxidation process of the heme-containing proteins, such as Hb and myoglobin.

Neuroglobin, an oxygen-binding protein in the mammalian nervous system (localization and putative functions)

The review summarizes current data on neuroglobin, the heme-containing protein discovered in mammalian nerve cells in 2000. It presents general characteristics of neuroglobin as well as data on its evolutionary changes and expression across different taxa. Neuroglobin distribution in specific brain structures and outside the brain is described. The issue of the occurrence of neuroglobin not only in neurons but also in astroglial cells is discussed. Subcellular localization of neuroglobin is characterized with a special focus on its detection in the nucleus of nerve cells, suggesting its involvement in nuclear functions. Current ideas on the probable functional significance of neuroglobin are reported. Neuroglobin is presumed to be involved in metabolism of reactive nitrogen and oxygen species as well as in intracellular signaling pathways. Besides, neuroglobin has neuroprotective and antiapoptotic functions. Since its expression changes during ontogenesis, its neuroprotective role in ageing is specifically highlighted. Changes in expression and localization of neuroglobin are suggested to influence the adaptive potential of an organism.

P 008 - Formation of cyanogen iodide by heme peroxidases

The heme-containing proteins lactoperoxidase (LPO) and myeloperoxidase are important enzymes involved in immune defence reactions. They are well known for their ability to oxidise in a hydrogen peroxide-driven reaction thiocyanate, iodide and in case of myeloperoxidase also bromide and chloride to the corresponding hypo(pseudo)halous acids. Here we describe the formation of cyanogen iodide (ICN) as a novel product of these enzymes. The formation of ICN was evidenced by both 13 C-nuclear magnetic resonance spectroscopy by applying 13 C-labeled thiocyanate and by head-space gas-chromatography mass spectrometry. This product is only formed when iodide is used in excess over thiocyanate during these reactions. Furthermore, the formation of bactericidal components by the LPO-hydrogen peroxide-thiocyanate/iodide system was investigated on a luminescent E. coli strain. This approach delivered time-resolved data about the killing of these microorganisms. In the mixed presence of thiocyanate and iodide (with an excess of iodide) a considerably higher killing rate was observed than in the sole application of either ion species. These data correspond well with the direct apllication of ICN. Control experiments of the determination of colony-forming units confirmed the results. Our data offer a novel product pathway that may be important for numerous biotechnological application of LPO.

Biomimetic behavior and nanomolar detection of hydrogen peroxide on an electrochemically pre-treated hematin modified glassy carbon electrode

An electrochemical pre-treatment of a glassy carbon electrode in sodium bicarbonate solution was performed to adsorb hematin (aHtin-GCE), an iron (III) porphyrin present in heme-containing proteins, in order to prepare a new biomimetic sensor for peroxidases. The aHtin-GCe presented peroxidase catalytic activity and a biomimetic behavior related to enzymes responsible for the reduction of H2O2. The previous glassy carbon surface functionalization in bicarbonate solution also proved to be an essential parameter to be controlled as it provides carboxylic acids terminations, which stabilizes the hematin molecule on the electrode surface. The aHtin-GCE kinetics fitted with the Michaelis–Menten model assume the formation of an enzyme-substrate complex in which H2O2 bonds to the electrochemically reduced iron(II) center with further reduction of the peroxide to peroxyl radical. The observation that lithium ions act as competitive inhibitors toward H2O2 also provided the insight toward the application of the aHtin-GCE as a model dispositive to evaluate peroxidase inhibitors. The aHtin-GCE peak current presented a linear correlation with the concentration of H2O2 from the 400nM to 800nM range with an experimental LoD of 0.11μM.

Functional roles of globin proteins in hypoxia-tolerant ectothermic vertebrates.

Globins are heme-containing proteins ubiquitously expressed in vertebrates, where they serve a broad range of biological functions, directly or indirectly related to the tight control of oxygen levels and its toxic products in vivo. Perhaps the most investigated of all proteins, hemoglobin and myoglobin are primarily involved in oxygen transport and storage, but also in facilitating arterial vasodilation, suppressing mitochondrial respiration, and preventing tissue oxidative damage via accessory redox enzymatic activities during hypoxia. By contrast, the more recently discovered neuroglobin and cytoglobin do not seem to function as reversible oxygen carriers and are instead involved in redox activities, although their exact biological roles remain to be clarified. In this context, hypoxia-tolerant ectotherms, such as freshwater turtles and members of the carp family that survive winter in extreme hypoxia, have proven as excellent models to appreciate the diversity of biological functions of globin proteins. Unraveling physiological roles of globin proteins in these extreme animals will clarify an important part of the adaptive mechanisms for surviving extreme fluctuations of oxygen availability that are prohibitive to mammals.

Heme-Based Oxygen Sensor Histidine Kinase Afgchk and Its Intra- and Interprotein Signal Transduction Observed By Hydrogen/Deuterium Exchange and Crystallography

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 (the intraprotein or interdomain signal transduction). Subsequently, this phosphate is then transferred to Asp52 or Asp169 of the RR protein (interprotein or protein-protein signal transduction). Several biochemical approaches were utilized in order to study both the intra- and interprotein signal transduction of AfGcHK and its two-component system: (i) enzyme kinetic study, (ii) hydrogen/deuterium exchange experiments associated with mass spectrometry, (iii) cross-linking studies, (iv) analytical ultracentrifugation and (v) X-ray crystallography. Regarding the interprotein signal transduction we have found that AfGcHK and the RR protein form a complex with 2:1 stoichiometry utilizing the analytical ultracentrifugation. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) studies on the AfGcHK:RR complex showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein’s Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Our results on intraprotein signal transduction indicate that the coordination and oxidation state of the sensor domain’s heme iron center profoundly affect the functional domain’s catalytic activity because they modulate its ATP affinity and thus change its k cat/K m ATPvalue. The contact area between the sensor and functional domains was identified by hydrogen/deuterium exchange experiments in conjunction with mass spectrometry and cross-linking studies. Moreover, X-ray crystallography revealed that the structure of the sensor domain when the heme iron complex is in the ferric state (which is associated with an active functional domain) differs from that when the complex is in the ferrous state (which is associated with an inactive functional domain). All the above mentioned findings will be discussed to illustrate the mechanism of both intra- and interprotein signal transduction in this globin-coupled histidine kinase as a representative of heme-containing oxygen sensor proteins in general.