Globin phylogeny, evolution and function, the newest update.

Our globin census update allows us to refine our vision of globin origin, evolution, and structure to function relationship in the context of the currently accepted tree of life. The modern globin domain originates as a single domain, three-over-three α-helical folded structure before the diversification of the kingdoms of life (Bacteria, Archaea, Eukarya). Together with the diversification of prokaryotes, three monophyletic globin families (M, S, and T) emerged, most likely in Proteobacteria and Actinobacteria, displaying specific sequence and structural features, and spread by vertical and horizontal gene transfer, most probably already present in the last universal common ancestor (LUCA). Non-globin domains were added, and eventually lost again, creating multi-domain structures in key branches of M- (FHb and Adgb) and the vast majority of S globins, which with their coevolved multi-domain architectures, have predominantly "sensor" functions. Single domain T-family globins diverged into four major groups and most likely display functions related to reactive nitrogen and oxygen species (RNOS) chemistry, as well as oxygen storage/transport which drives the evolution of its major branches with their characteristic key distal residues (B10, E11, E7, and G8). M-family evolution also lead to distinctive major types (FHb and Fgb, Ngb, Adgb, GbX vertebrate Gbs), and shows the shift from high oxygen affinity controlled by TyrB10-Gln/AsnE11 likely related to RNOS chemistry in microorganisms, to a moderate oxygen affinity storage/transport function controlled by hydrophobic B10/E11-HisE7 in multicellular animals.

Hell's Gate Globin-I from Methylacidiphilum infernorum Displays a Unique Temperature-Independent pH Sensing Mechanism Utililized a Lipid-Induced Conformational Change.

Hell's Gate globin-I (HGb-I) is a thermally stable globin from the aerobic methanotroph Methylacidiphilium infernorum. Here we report that HGb-I interacts with lipids stoichiometrically to induce structural changes in the heme pocket, changing the heme iron distal ligation coordination from hexacoordinate to pentacoordinate. Such changes in heme geometry have only been previously reported for cytochrome c and cytoglobin, linked to apoptosis regulation and enhanced lipid peroxidation activity, respectively. However, unlike cytoglobin and cytochrome c, the heme iron of HGb-I is altered by lipids in ferrous as well as ferric oxidation states. The apparent affinity for lipids in this thermally stable globin is highly pH-dependent but essentially temperature-independent within the range of 20-60 °C. We propose a mechanism to explain these observations, in which lipid binding and stability of the distal endogenous ligand are juxtaposed as a function of temperature. Additionally, we propose that these coupled equilibria may constitute a mechanism through which this acidophilic thermophile senses the pH of its environment.

Architectural digest: Thermodynamic stability and domain structure of a consensus monomeric globin.

Artificial proteins representing the consensus of a set of homologous sequences have attracted attention for their increased thermodynamic stability and conserved activity. Here, we applied the consensus approach to a b-type heme-binding protein to inspect the contribution of a dissociable cofactor to enhanced stability and the chemical consequences of creating a generic heme environment. We targeted the group 1 truncated hemoglobin (TrHb1) subfamily of proteins for their small size (∼120 residues) and ease of characterization. The primary structure, derived from a curated set of ∼300 representative sequences, yielded a highly soluble consensus globin (cGlbN) enriched in acidic residues. Optical and NMR spectroscopies revealed high-affinity heme binding in the expected site and in two orientations. At neutral pH, proximal and distal iron coordination was achieved with a pair of histidine residues, as observed in some natural TrHb1s, and with labile ligation on the distal side. As opposed to studied TrHb1s, which undergo additional folding upon heme binding, cGlbN displayed the same extent of secondary structure whether the heme was associated with the protein or not. Denaturation required guanidine hydrochloride and showed that apo- and holoprotein unfolded in two transitions-the first (occurring with a midpoint of ∼2 M) was shifted to higher denaturant concentration in the holoprotein (∼3.7 M) and reflected stabilization due to heme binding, while the second transition (∼6.2 M) was common to both forms. Thus, the consensus sequence stabilized the protein but exposed the existence of two separately cooperative subdomains within the globin architecture, masked as one single domain in TrHb1s with typical stabilities. The results suggested ways in which specific chemical or thermodynamic features may be controlled in artificial heme proteins.

New Aminobiphenylcysteine Derivatives in Globin and Urine of Rats Dosed with 4-Aminobiphenyl, a Tobacco Smoke Carcinogen.

The 4-biphenylnitrenium ion (BPN), a reactive metabolic intermediate of the tobacco smoke carcinogen 4-aminobiphenyl (4-ABP), can react with nucleophilic sulfanyl groups in glutathione (GSH) as well as in proteins. The main site of attack of these S-nucleophiles was predicted using simple orientational rules of aromatic nucleophilic substitution. Thereafter, a series of presumptive 4-ABP metabolites and adducts with cysteine were synthesized, namely, S-(4-amino-3-biphenyl)cysteine (ABPC), N-acetyl-S-(4-amino-3-biphenyl)cysteine (4-amino-3-biphenylmercapturic acid, ABPMA), S-(4-acetamido-3-biphenyl)cysteine (AcABPC), and N-acetyl-S-(4-acetamido-3-biphenyl)cysteine (4-acetamido-3-biphenylmercapturic acid, AcABPMA). Then, globin and urine of rats dosed with a single ip dose of 4-ABP (27 mg/kg b.w.) was analyzed by HPLC-ESI-MS2. ABPC was identified in acid-hydrolyzed globin at levels of 3.52 ± 0.50, 2.74 ± 0.51, and 1.25 ± 0.12 nmol/g globin (mean ± S.D.; n = 6) on days 1, 3, and 8 after dosing, respectively. In the urine collected on day 1 (0-24 h) after dosing, excretion of ABPMA, AcABPMA, and AcABPC amounted to 1.97 ± 0.88, 3.09 ± 0.75, and 3.69 ± 1.49 nmol/kg b.w. (mean ± S.D.; n = 6), respectively. On day 2, excretion of the metabolites decreased by one order of magnitude followed by a slower decrease on day 8. Regarding the possible formation of AcABPC from ABPC, N-acetylation of the amino group at the biphenyl moiety prior to that at cysteine appears to be very unlikely. Thus, the structure of AcABPC indicates the involvement of N-acetyl-4-biphenylnitrenium ion (AcBPN) and/or its reactive ester precursors in in vivo reactions with GSH and protein-bound cysteine. ABPC in globin might become an alternative biomarker of the dose of toxicologically relevant metabolic intermediates of 4-ABP.

The X-ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand-binding sites.

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

Evolutionary analysis of globin domains from kinetoplastids.

Globin (Gb) domains function in sensing gaseous ligands like oxygen and nitric oxide. In recent years, Gb domain containing heme binding adenylate cyclases (OsAC or GbAC) emerged as significant modulator of Leishmania response to hypoxia and oxidative stress. During progression of life cycle stages, kinetoplastids experience altered condition in insect vectors or other hosts. Moreover, marked diversity in life style has been accounted among kinetoplastids. Distribution and abundance of Gb-domains vary between different groups of kinetoplastids. While in bodonoids, Gbs are not combined with any other functional domains, in trypanosomatids it is either fused with adenylate cyclase (AC) or oxidoreductase (OxR) domains. In salivarian trypanosomatids and Leishmania (Viannia) subtypes, no gene product featuring Gbs can be identified. In this context, evolution of Gb-domains in kinetoplastids was explored. GbOxR derived Gbs clustered with bacterial flavohemoglobins (fHb) including one fHb from Advenella, an endosymbiont of monoxeneous trypanosomatids. Codon adaptation and other evolutionary analysis suggested that OsAC (LmjF.28.0090), the solitary Gb-domain featuring gene product in Leishmania, was acquired via possible horizontal gene transfer. Substantial functional divergence was estimated between orthologues of genes encoding GbAC or GbOxR; an observation also reflected in structural alignment and heme-binding residue predictions. Orthologue-paralogue and synteny analysis indicated genomic reduction in GbOxR and GbAC loci for dixeneous trypanosomatids.

Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor AfGcHK abolishes bacterial signal transduction.

The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

Recent Insights into the Diversity and Evolution of Invertebrate Hemerythrins and Extracellular Globins.

There are three broad groups of oxygen-transport proteins found in the haemolymph (blood) of invertebrates, namely the hemocyanins, the hemerythrins and the globins. Both hemerythrins and extracellular globins are iron-based proteins that are understudied when compared to the copper-containing hemocyanins. Recent evidence suggests that hemerythrins and (giant) extracellular globins (and their linker chains) are more widely distributed than previously thought and may have biological functions beyond oxygen transport and storage. Herein, we review contemporary literature of these often-neglected proteins with respect to their structural configurations on formation and ancestral states.

Understanding contact patterns of protein structures from protein contact map and investigation of unique patterns in the globin-like folded domains.

Proteins are biochemical compounds made up of one or more polypeptides in a specific order, typically folded into a functionally active form. Proteins are categorized into four different structural classes according to the topology of α-helices and ß-strands. In this study, we modeled these four structural classes as an undirected network depicting amino acids as nodes and interaction between them as edges. Results infer that basic protein classes can be easily recognized as well as distinguished by utilizing protein contact maps (PCM). Toward studying the globin-like fold, the helix-loop-helix region contacts were seen to be of a unique pattern, and these remained in all the folds. Further, the averaged diagonal contacts were analyzed and identified those contacts in α/ß proteins were higher in comparison with the other class. Interesting, we noticed that anti-parallel beta sheets were dominant in all-ß and α + ß classes that lead to similar diagonal patterns. Network properties of all four basic classes were analyzed and found to possess small-world property. Findings infer that PCM may assist classify protein structure classes and it also helps in evaluating the predicted protein structures.

Thermal Stability of Globins: Implications of Flexibility and Heme Coordination Studied by Molecular Dynamics Simulations.

Proteins are sensitive to temperature, and abrupt changes in the normal temperature conditions can have a profound impact on both structure and function, leading to protein unfolding. However, the adaptation of certain organisms to extreme conditions raises questions about the structural features that permit the structure and function of proteins to be preserved under these adverse conditions. To gain insight into the molecular basis of protein thermostability in the globin family, we have examined three representative examples: human neuroglobin, horse heart myoglobin, and Drosophila hemoglobin, which differ in their melting temperatures and coordination states of the heme iron in the absence of external ligands. In order to elucidate the possible mechanisms that govern the thermostability of these proteins, microsecond-scale classical molecular dynamics simulations were performed at different temperatures. Structural fluctuations and essential dynamics were analyzed, indicating that the flexibility of the CD region, which includes the two short C and D helixes and the connecting CD loop, is directly related to the thermostability. We observed that a larger inherent flexibility of the protein produces higher thermostability, probably concentrating the thermal fluctuations observed at high temperature in flexible regions, preventing unfolding. Globally, the results of this work improve our understanding of thermostability in the globin family.

Pyrrole adducts in globin and plasma of workers exposed to hexane.

OBJECTIVES: Urinary excretion of 2,5-hexanedione is currently used to estimate the exposure levels of hexane occurring to an individual during the previous work shift. However, because hexane exposures and urinary 2,5-hexanedione levels can vary considerably from day to day, and subchronic to chronic exposures to hexane are required to produce neuropathy, this biomarker may not accurately reflect the risk of an individual for developing hexane neuropathy. This investigation examines the potential of hexane-derived pyrrole adducts produced on globin and plasma proteins as markers for integrating cumulative exposures. Because the pyrrole markers incorporate bioactivation of hexane to 2,5-hexandione and the initial step of protein adduction involved in hexane-induced neuropathy, they potentially can serve as biomarkers of effect through reflecting pathogenetic events within the nervous system. Additionally, pyrrole formation is an irreversible reaction suggesting that hexane-derived protein pyrroles can be used to assess cumulative exposures to provide a better characterization of individual susceptibilities. METHODS: To examine the utility of the proposed markers, blood samples were obtained from eleven workers who used hexane for granulating metal powders in a slurry to produce metal machining die tools and four non-exposed volunteers. Globin and plasma were isolated, and the proteins were digested using pepsin, reacted with Ehrlich's reagent and the level of pyrrole adducts were determined by absorbance at 530 nm. To determine the dose-response curve and dynamic range of the assay, erythrocytes were incubated with a range of 2,5-hexanedione concentrations and the net absorbance at 530 nm of isolated globin was measured. RESULTS: Pyrrole was detected in both the globin and plasma samples of the workers exposed to hexane and the levels of pyrroles in plasma were positively correlated with the levels of pyrroles in globin for most of the workers. CONCLUSIONS: This investigation demonstrates that detectable levels of hexane-derived protein pyrrole adducts are produced on peripheral proteins following occupational exposures to hexane and supports the utility of measuring pyrroles for integrating cumulative exposures to hexane.

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin.

Hydrogen sulfide is a critical signaling molecule, but high concentrations cause cellular toxicity. A four-enzyme pathway in the mitochondrion detoxifies H2S by converting it to thiosulfate and sulfate. Recent studies have shown that globins like hemoglobin and myoglobin can also oxidize H2S to thiosulfate and hydropolysulfides. Neuroglobin, a globin enriched in the brain, was reported to bind H2S tightly and was postulated to play a role in modulating neuronal sensitivity to H2S in conditions such as stroke. However, the H2S reactivity of the coordinately saturated heme in neuroglobin is expected a priori to be substantially lower than that of the 5-coordinate hemes present in myoglobin and hemoglobin. To resolve this discrepancy, we explored the role of the distal histidine residue in muting the reactivity of human neuroglobin toward H2S. Ferric neuroglobin is slowly reduced by H2S and catalyzes its inefficient oxidative conversion to thiosulfate. Mutation of the distal His64 residue to alanine promotes rapid binding of H2S and its efficient conversion to oxidized products. X-ray absorption, EPR, and resonance Raman spectroscopy highlight the chemically different reaction options influenced by the distal histidine ligand. This study provides mechanistic insights into how the distal heme ligand in neuroglobin caps its reactivity toward H2S and identifies by cryo-mass spectrometry a range of sulfide oxidation products with 2-6 catenated sulfur atoms with or without oxygen insertion, which accumulate in the absence of the His64 ligand.

Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

Photoinduced transport in an H64Q neuroglobin antidote for carbon monoxide poisoning.

Carbon monoxide (CO) is a leading cause of poisoning deaths worldwide, without available antidotal therapy. Recently, a potential treatment for CO poisoning was introduced, based on binding of CO by neuroglobin (Ngb) with a mutated distal histidine (H64Q). Here, we present an atomistic mechanism of CO trapping by H64Q Ngb revealed by nonadiabatic molecular dynamics. We focused on CO photodissociation and recombination of CO to wild type (WT) and H64Q Ngb. Our results demonstrate that the distribution of CO within the proteins differs substantially due to rearrangement of amino acids surrounding the distal heme pocket. This leads to the decrease of the distal pocket volume in H64Q Ngb in comparison to WT Ngb, trapping migrating CO molecules in the distal pocket. We show that the mutation implicates the shortening of the time scale of CO geminate recombination, making H64Q Ngb 2.7 times more frequent binder than WT Ngb.

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78 mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30 g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

Mapping Hydrophobic Tunnels and Cavities in Neuroglobin with Noble Gas under Pressure.

Internal cavities are crucial for conformational flexibility of proteins and can be mapped through noble gas diffusion and docking. Here we investigate the hydrophobic cavities and tunnel network in neuroglobin (Ngb), a hexacoordinated heme protein likely to be involved in neuroprotection, using crystallography under noble gas pressure, mostly at room temperature. In murine Ngb, a large internal cavity is involved in the heme sliding mechanism to achieve binding of gaseous ligands through coordination to the heme iron. In this study, we report that noble gases are hosted by two major sites within the internal cavity. We propose that these cavities could store oxygen and allow its relay in the heme proximity, which could correspond to NO location in the nitrite-reductase function of Ngb. Thanks to a recently designed pressurization cell using krypton at high pressure, a new gas binding site has been characterized that reveals an alternate pathway for gaseous ligands. A new gas binding site on the proximal side of the heme has also been characterized, using xenon pressure on a Ngb mutant (V140W) that binds CO with a similar rate and affinity to the wild-type, despite a reshaping of the internal cavity. Moreover, this study, to our knowledge, provides new insights into the determinants of the heme sliding mechanism, suggesting that the shift at the beginning of helix G precedes and drives this process.

Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases.

Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. Significance statement: Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our findings demonstrate that the inhibition of sGCs by neuroglobin is essential for rapid adaptation to either low or high oxygen levels, and that similar structural regions are key for regulating both oxygen and nitric oxide sensors. Based on our structural and functional analyses, we present the hypothesis that neuroglobin-sGC interactions may be generally important for adaptation processes, including those in organisms with more complex neurological functions.

Efficient Reduction of Vertebrate Cytoglobins by the Cytochrome b5/Cytochrome b5 Reductase/NADH System.

Cytoglobin is a heme-containing protein ubiquitous in mammalian tissues. Unlike the evolutionarily related proteins hemoglobin and myoglobin, cytoglobin shows a six-coordinated heme binding, with the heme iron coordinated by two histidine side chains. Cytoglobin is involved in cytoprotection pathways through yet undefined mechanisms, and it has recently been demonstrated that cytoglobin has redox signaling properties via nitric oxide (NO) and nitrite metabolism. The reduced, ferrous cytoglobin can bind oxygen and will react with NO in a dioxygenation reaction to form nitrate, which dampens NO signaling. When deoxygenated, cytoglobin can bind nitrite and reduce it to NO. This oxidoreductase activity could be catalytic if an effective reduction system exists to regenerate the reduced heme species. The nature of the physiological cytoglobin reducing system is unknown, although it has been proposed that ascorbate and cytochrome b5 could fulfill this role. Here we describe that physiological concentrations of cytochrome b5 and cytochrome b5 reductase can reduce human and fish cytoglobins at rates up to 250-fold higher than those reported for their known physiological substrates, hemoglobin and myoglobin, and up to 100-fold faster than 5 mM ascorbate. These data suggest that the cytochrome b5/cytochrome b5 reductase system is a viable reductant for cytoglobin in vivo, allowing for catalytic oxidoreductase activity.

Electrochemical Evidence for Neuroglobin Activity on NO at Physiological Concentrations.

The true function of neuroglobin (Ngb) and, particularly, human Ngb (NGB) has been under debate since its discovery 15 years ago. It has been expected to play a role in oxygen binding/supply, but a variety of other functions have been put forward, including NO dioxygenase activity. However, in vitro studies that could unravel these potential roles have been hampered by the lack of an Ngb-specific reductase. In this work, we used electrochemical measurements to investigate the role of an intermittent internal disulfide bridge in determining NO oxidation kinetics at physiological NO concentrations. The use of a polarized electrode to efficiently interconvert the ferric (Fe(3+)) and ferrous (Fe(2+)) forms of an immobilized NGB showed that the disulfide bridge both defines the kinetics of NO dioxygenase activity and regulates appearance of the free ferrous deoxy-NGB, which is the redox active form of the protein in contrast to oxy-NGB. Our studies further identified a role for the distal histidine, interacting with the hexacoordinated iron atom of the heme, in oxidation kinetics. These findings may be relevant in vivo, for example, in blocking apoptosis by reduction of ferric cytochrome c, and gentle tuning of NO concentration in the tissues.

Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.