Binding of Ferric Heme by the Recombinant Globin from the Cyanobacterium <i>Synechocystis</i> sp. PCC 6803

Jt, Lecomte; Nl, Scott; Bc, Vu; Cj, Falzone

doi:10.1021/bi010226u

Cited by 47 publications

(85 citation statements)

References 78 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As mentioned above, Ss-2/2HbN displays bis-histidine heme hexacoordination, where HisF8 and HisE10 are the Fe-atom ligands (21,22,32). Binding of an exogenous ligand to the heme distal site, requires the dissociation of HisE10 from the heme and remarkable conformational changes affecting the B-and E-helices (12,33).…”

Section: Ligand Binding At the Heme Distal Sitementioning

confidence: 95%

Protein structure in the truncated (2/2) hemoglobin family

Pesce¹,

Nardini

Milani

et al. 2007

IUBMB Life

View full text Add to dashboard Cite

The discovery of protein sequences belonging to the widespread 'truncated hemoglobin' family has been followed in the last few years by extensive analyses of their three‐dimensional structures. Truncated hemoglobins can be classified in three main groups, in light of their overall structural properties. The three groups adopt a 2‐on‐2 α‐helical sandwich fold, based on four main α‐helices of the classical 3‐on‐3 α‐helical sandwich found in vertebrate and invertebrate globins. Each of the three groups displays sequence and structure specific features. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and residue TyrB10 is almost invariant in the three groups. Despite sequence variability in the heme distal site region, a strongly intertwined, but varied, network of hydrogen bonds stabilizes the heme ligand in the three protein groups. Fine mechanisms of ligand recognition and stabilization may vary based on group‐specific distal site residues and on differing ligand diffusion pathways to the heme. Taken together, the structural considerations here presented underline that 'truncated hemoglobins' result from careful editing of the 3‐on‐3 α‐helical globin sandwich fold, rather than from simple 'truncation' events. Thus, '2/2Hb' appears the most proper term to concisely address this protein family.

show abstract

Section: Ligand Binding At the Heme Distal Sitementioning

confidence: 95%

Protein structure in the truncated (2/2) hemoglobin family

Pesce¹,

Nardini

Milani

et al. 2007

IUBMB Life

View full text Add to dashboard Cite

show abstract

“…The formation of this complex inhibits ␣Hb peroxidase activity and heme loss (2). Bishistidyl heme coordination is becoming increasingly recognized as a feature of numerous vertebrate and non-vertebrate globins (14) and has been shown previously to confer a relative stabilization of the Fe 3ϩ over the Fe 2ϩ oxidation state (15)(16)(17). Although bis-histidyl heme coordination has previously been detected in solutions of met-Hb, formed through spontaneous autoxidation of Hb (18 -21), the bishis-␣Hb⅐AHSP complex provides the first evidence that the bis-histidyl heme may play a positive functional role in Hb biochemistry by inhibiting the production of harmful reactive oxygen species.…”

mentioning

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

View full text Add to dashboard Cite

␣-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...

show abstract

“…NOEs between the heme 8-CH 3 and Tyr65, heme 2-vinyl and Phe84, heme 2-vinyl and Tyr53, heme 4-vinyl and Phe34, heme 3-CH 3 and Leu79, and heme 3-CH 3 and Val87 all confirmed similar placement of the heme. The relative proportions of native protein populating two states that differ by a 180°r otation about the heme α-γ meso axis is 95:5 in the wild-type hemichrome (4). The ratio shifted slightly in favor of the minor form in the variant.…”

Section: Characterization Of A69s S6803 Rhb-r In the Ferric Statementioning

confidence: 93%

“…The parameters for the NMR experiments (acquisition and processing) have been reported elsewhere (4). NMR data were processed with NMRpipe (44) or XWIN-NMR (Bruker BioSpin).…”

Section: Nmr Spectroscopymentioning

confidence: 99%

“…Absorbance data (412 and 370 nm) and CD data (209 and 222 nm) were analyzed with Savuka to obtain the free energy of unfolding and the dependence of ΔG° on denaturant concentration [D]. A linear dependence was assumed: (4) . Although evidence was found for the population of at least one equilibrium unfolding intermediate (see results), individual denaturation curves were fit satisfactorily with a two-state model.…”

Section: Chemical Denaturationmentioning

confidence: 99%

See 1 more Smart Citation

Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin

Knappenberger

Kuriakose

et al. 2006

Biochemistry

Self Cite

View full text Add to dashboard Cite

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O 2 , CO, and CN − , among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine whereas, in myoglobins, it is a serine involved in an intricate hydrogen bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala → Ser replacement. The substitution resulted in minor structural perturbations, but the holoprotein's response to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased two-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.Keywords truncated hemoglobin; 2-on-2 globin; hexacoordinate hemoglobin; urea-gradient gel; heme binding; thermodynamic stabilityIron protoporphyrin IX (Fe-PPIX) 1 serves as a cofactor in a large number of essential proteins. In the absence of covalent attachment to the protein matrix via protoporphyrin substituents, the contact between Fe(II)-PPIX (or b heme) and the protein is limited to ligation bond(s) to the iron, hydrogen bonding, van der Waals and electrostatic interactions, and hydrophobic † This study was supported by National Science Foundation grants MCB-091182 and MCB-0349409, National Institutes of Health grant GM-054217, and NASA grant NNG04GN33H (DAV).* To whom correspondence should be addressed. Tel: (814) NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 11. forces. The affinity for the heme group and its chemical properties, such as redox potential, ability to bind various ligands, and propensity for generating reactive oxygen species, are strictly controlled by the protein environment.In many b hemoproteins the iron is endogenously hexacoordinated, with the axial positions of its octahedral geometry filled by two histidine side chains (1). A well-known example is cytochrome b 5 , an electron-transfer protein that does not bind...

show abstract

Binding of Ferric Heme by the Recombinant Globin from the Cyanobacterium Synechocystis sp. PCC 6803

Cited by 47 publications

References 78 publications

Protein structure in the truncated (2/2) hemoglobin family

Protein structure in the truncated (2/2) hemoglobin family

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

Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin

Contact Info

Product

Resources

About