The crystal structure of a tetrameric hemoglobin in a partial hemichrome state

Riccio, Antonio; Vitagliano, Luigi; Prisco, Guido di; Zagari, Adriana; Mazzarella, L.

doi:10.1073/pnas.132182099

Cited by 74 publications

(103 citation statements)

References 38 publications

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“…Defining the role of bis-histidyl heme coordination in erythroid biology should also provide insights into the functional properties of bis-histidyl heme structures in other systems, such as the hemoglobins of some arctic fish species (38) and other mammalian globins such as neuroglobin and cytoglobin (39). Ultimately, understanding how and why AHSP promotes formation of bis-histidyl ␣Hb may allow us to design experimental strategies to modify AHSP function in new treatments for thalassemia (40).…”

Section: Discussionmentioning

confidence: 99%

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Gell

Feng

Zhou

et al. 2009

Journal of Biological Chemistry

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␣-Hemoglobin (␣Hb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with ␣Hb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting ␣Hb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in ␣Hb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro 30 , in loop 1 of AHSP. Mutation of Pro 30 to a variety of residue types results in reduced ability to convert ␣Hb. In complex with ␣Hb, AHSP Pro 30 adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in ␣Hb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the ␣Hb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of ␣Hb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA) 5 is a tetramer of two ␣Hb and two ␤Hb subunits, which is produced to extremely high concentrations (ϳ340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified ␣-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with ␣Hb (see Fig. 1A) (2) but does not interact with ␤Hb or HbA. AHSP also binds heme-free (apo) ␣Hb (3) and may serve functions in both the folding of nascent ␣Hb (4) and the detoxification of excess ␣Hb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in ␤-thalassemia patients due to the cytotoxic effects of free ␣Hb.Free ␣Hb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7,8). Reactive oxygen species can damage heme, ␣Hb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9 -12). The presence of AHSP may explain how cells tolerate the slight excess of ␣Hb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional ␤Hb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies ␣Hb. AHSP binds to oxygenated ␣Hb to generate an initial complex that retains the oxy-hem...

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Section: Discussionmentioning

confidence: 99%

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Gell

Feng

Zhou

et al. 2009

Journal of Biological Chemistry

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“…A tetrameric Hb in a partial hemichrome state was first observed by some of the present authors while investigating airoxidized crystals of the main Hb component isolated from the Antarctic fish Trematomus newnesi (Hb1Tn) (23). Successively, the formation and stability of hemichrome species in solution, as well as their structural features in the solid state were extensively investigated for several Antarctic fish Hbs.…”

Section: Occurrence Of Bis-histidyl Adductsmentioning

confidence: 70%

“…For tetrameric Hbs of Antarctic fish, interesting observations have been obtained regarding the quaternary-structure (8,23,24,29). These Hbs form only partial hemichrome states either in the a or in the b chain, strongly supporting the intrinsic differences between the two chains in terms of backbone flexibility.…”

Section: Structural Featuresmentioning

confidence: 87%

“…To date, only a few Hb structures with bis-histidyl endogenous coordination have been deposited in the PDB (10,(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

Section: Occurrence Of Bis-histidyl Adductsmentioning

confidence: 99%

“…These Hbs form only partial hemichrome states either in the a or in the b chain, strongly supporting the intrinsic differences between the two chains in terms of backbone flexibility. Interestingly, while in horse Hb, the hemichrome is formed at the a heme (24), in Antarctic fish Hbs (HbTb and Hb1Tn) only the b heme is involved (8,23,29). As the quaternary assembly is very similar, these findings suggest a change in the relative flexibility of the two chains.…”

Section: Structural Featuresmentioning

confidence: 99%

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Occurrence and formation of endogenous histidine hexa‐coordination in cold‐adapted hemoglobins

et al. 2011

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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 β 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 α and β 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

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Combined crystallographic and spectroscopic analysis of Trematomus bernacchii hemoglobin highlights analogies and differences in the peculiar oxidation pathway of Antarctic fish hemoglobins

et al. 2009

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Recent studies have demonstrated that hemoglobins isolated from Antarctic fish undergo peculiar oxidation processes. Here we show, by combining crystallographic and spectroscopic data, that the oxidation pathway of Trematomus bernacchii hemoglobin (HbTb) is distinct from that observed for the major component of Trematomus newnesi (Hb1Tn), despite the high sequence identity of the two proteins and structural similarity of their ferrous and fully oxidized states. Resonance Raman analysis of HbTb autoxidation upon air-exposure reveals the absence of the oxidized pentacoordinated state that was observed for Hb1Tn. The HbTb oxidation pathway is characterized by two ferric species: an aquo hexacoordinated high spin state and a bis-histidyl hexacoordinated low spin form, which appear in the early stages of the oxidation process. The high resolution structure of an intermediate along the oxidation pathway has been determined at 1.4 A resolution. The analysis of the electron density of the heme pocket shows, for both the alpha and the beta iron, the coexistence of multiple binding states. In this partially oxidized form, HbTb exhibits significant deviations from the canonical R state both at the local and global level. The analysis of these modifications highlights the structural correlation between key functional regions of the protein.

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The crystal structure of a tetrameric hemoglobin in a partial hemichrome state

Cited by 74 publications

References 38 publications

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Occurrence and formation of endogenous histidine hexa‐coordination in cold‐adapted hemoglobins

Combined crystallographic and spectroscopic analysis of Trematomus bernacchii hemoglobin highlights analogies and differences in the peculiar oxidation pathway of Antarctic fish hemoglobins

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