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An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP: The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.

The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed. © 2011 IUBMB IUBMB Life, 63(7): 566–573, 2011

Spectroscopic and crystallographic evidence of endogenous (His) ligation at the sixth coordination site of the heme iron has been reported for monomeric, dimeric, and tetrameric hemoglobins (Hbs) in both ferrous (hemochrome) and ferric (hemichrome) oxidation states. In particular, the ferric bis- histidyl adduct represents a common accessible ordered state for the ² chains of all tetrameric Hbs isolated from Antarctic and sub-Antarctic fish. Indeed, the crystal structures of known tetrameric Hbs in the bis-His state are characterized by a different binding state of the ± and ² chains. An overall analysis of the bis-histidyl adduct of globin structures deposited in the Protein Data Bank reveals a marked difference between hemichromes in tetrameric Hbs compared to monomeric/dimeric Hbs. Herein, we review the structural, spectroscopic and stability features of hemichromes in tetrameric Antarctic fish Hbs. The role of bis-histidyl adducts is also addressed in a more evolutionary context alongside the concept of its potential physiological role.

The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three haemoglobins. The major haemoglobin (Hb1Em) displays the Root effect, a drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH. The carbomonoxy form of HbEm1 has been crystallized in two different crystal forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution diffraction data have been collected for both forms (1.45 and 1.49 Å resolution, respectively). The high-frequency resonance Raman spectra collected from the two crystal forms using excitation at 514 nm were almost indistinguishable. Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish haemoglobins.

Tetrameric hemoglobins represent the most commonly used model for the description of the basic concepts of protein allostery. The classical stereochemical model assumes a concerted transition of the protein, upon oxygen release, from the relaxed (R) to the tense (T) state. Despite the large amount of data accumulated on the end-points of the transition, scarce structural information is available on the intermediate species along the pathway. Here we report a spectroscopic characterization of the autoxidation process of the Trematomus newnesi major Hb component and the atomic resolution structure (1.25 A) of an intermediate form along the pathway characterized by a different binding and oxidation state of the alpha and beta chains. In contrast to the alpha-heme iron, which binds a CO molecule, the beta iron displays a pentacoordinated oxidized state, which is rare in tetrameric hemoglobins. Interestingly, the information provided by the present analysis is not limited to the characterization of the peculiar oxidation process of Antarctic fish hemoglobins. Indeed, this structure represents the most detailed snapshot of hemoglobin allosteric transition hitherto achieved. Upon ligand release at the beta heme, a cascade of structural events is observed. Notably, several structural features of the tertiary structure of the alpha and beta chains closely resemble those typically observed in the deoxygenated state. The overall quaternary structure also becomes intermediate between the R and the T state. The analysis of the alterations induced by the ligand release provides a clear picture of the temporal sequence of the events associated with the transition. The implications of the present findings have also been discussed in the wider context of tetrameric Hbs.

etrameric hemoglobins represent the most commonly used model for the description of the basic concepts of protein allostery. The classical stereochemical model assumes a concerted transition of the protein, upon oxygen release, from the relaxed (R) to the tense (T) state. Despite the large amount of data accumulated on the end-points of the transition, scarce structural information is available on the intermediate species along the pathway. Here we report a spectroscopic characterization of the autoxidation process of the Trematomus newnesi major Hb component and the atomic resolution structure (1.25 A) of an intermediate form along the pathway characterized by a different binding and oxidation state of the alpha and beta chains. In contrast to the alpha-heme iron, which binds a CO molecule, the beta iron displays a pentacoordinated oxidized state, which is rare in tetrameric hemoglobins. Interestingly, the information provided by the present analysis is not limited to the characterization of the peculiar oxidation process of Antarctic fish hemoglobins. Indeed, this structure represents the most detailed snapshot of hemoglobin allosteric transition hitherto achieved. Upon ligand release at the beta heme, a cascade of structural events is observed. Notably, several structural features of the tertiary structure of the alpha and beta chains closely resemble those typically observed in the deoxygenated state. The overall quaternary structure also becomes intermediate between the R and the T state. The analysis of the alterations induced by the ligand release provides a clear picture of the temporal sequence of the events associated with the transition. The implications of the present findings have also been discussed in the wider context of tetrameric Hbs.

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

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.

The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three haemoglobins. The major haemoglobin (Hb1Em) displays the Root effect, a drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH. The carbomonoxy form of HbEm1 has been crystallized in two different crystal forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution diffraction data have been collected for both forms (1.45 and 1.49 A ˚ resolution, respectively). The high-frequency resonance Raman spectra collected from the two crystal forms using excitation at 514 nm were almost indistinguishable. Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish haemoglobins.

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO(3)(-) catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 degrees C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO(2); the values of the second order catalytic rate constant (k(on)) are 4.1 x 10(5) and 4.5 x 10(5) m(-1) s(-1), respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k(on) (pK(a) = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO(2). The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO(2), ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 x 10(-4) and 9.7 x 10(-4) m. Under conditions where [ibuprofen] isL, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.