Spin relaxation of iron in mixed state hemoproteins.

In hemoproteins the relaxation mechanism of iron is Orbach for high spin (HS) and Raman for low spin (LS). We found that in met-hemoglobin and met-myoglobin, under conditions in which the two spin states coexist, both the HS and the LS states relax to the lattice through Orbach-like processes. Alos, very short (approximately 1 ns) and temperature independent transverse relaxation times T2 were estimated. This may result from the unusual electronic structure of mixed states hemoproteins that allows thermal equilibrium and interconversion of the spin states.     

Can Gas Replace Protein Function? CO Abrogates the Oxidative Toxicity of Myoglobin

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N₂. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.     

Haptoglobin phenotypes differ in their ability to inhibit heme transfer from hemoglobin to LDL

LDL oxidation plays a pivotal role in atherosclerosis. Excellular hemoglobin (Hb) is a trigger of LDL oxidation. By virtue of its ability to bind hemoglobin, haptoglobin (Hp) serves as an antioxidant. Oxidation of LDL by hemoglobin was analyzed to occur by heme displacement from methemoglobin lodged in LDL. The LDL-associated heme is disintegrated, and iron inserted this way in LDL triggers formation of lipid peroxides. The genetic polymorphism of haptoglobin was found to be a risk factor in the pathogenesis of atherosclerosis. Individuals with Hp2-2 have more vascular incidences as compared to those with Hp1-1. In the current study, oxidation of LDL by metHb was carried out at physiological pH without addition of external peroxides. Hb-derived oxidation of lipids and protein was found to be practically inhibited by Hp1-1 but only partially by Hp2-2. Heme transfer from metHb to LDL was almost completely omitted by Hp1-1 and only partially by Hp2-2. We concluded that partial heme transfer from the Hb-Hp2-2 complex to LDL is the reason for oxidation of LDL lipids as well as protein. These findings provide a molecular basis for Hp2-2 atherogenic properties.     

Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.     

A protein with multiple heme-binding sites from rabbit serum

A 93,000 molecular weight protein (HBP.93) which binds hemin and protoporphyrin IX with high affinity has been isolated from rabbit serum using affinity chromatography on hemin-conjugated agarose. The amino acid composition of this protein is unique in that the proline and histidine contents are remarkably high (16.6 and 9.9 mol %, respectively). A large increase in the absorbance of the Soret region arises from the heme-protein interaction. The spectrophotometric titration showed that the protein can bind 25-35 mol of hemin/mol of protein. The apparent dissociation constant was estimated to be 1-4 X 10(-7) M for hemin at pH 7.4 and approximately 10(-6) M for protoporphyrin IX at pH 9.2. The similarity of the difference spectrum of heme-HBP.93 complex to that of heme-hemopexin complex suggests that a bisimidazol-type coordination of heme iron is involved in the binding. The extremely high capacity of HBP.93 to bind heme is also demonstrated by a large increase in the sedimentation velocity of the protein upon heme binding. The native heme-protein complex migrates faster than the heme-free protein in a polyacrylamide gel at pH 8.8; the increased mobility appears to be due to the charge on the carboxyl groups of the bound heme. Although the use of a hemin-agarose column has failed to reveal a protein of similar size and heme affinity in the sera of a number of other species, including man, the heme-binding properties and high histidine level of the human alpha 2-histidine-rich glycoprotein raise the possibility that the two proteins are related.     

Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (beta-hematin formation)

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (beta-hematin formation) through its oxidation. Heme when added to HRP compound II (FeIV=O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, FeIII) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1H=14.75 G, a2H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.     

The reactivity of thiols and disulfides with different redox states of myoglobin. Redox and addition reactions and formation of thiyl radical intermediates.

The reactivity of several thiols, including glutathione, dihydrolipoic acid, cysteine, N-acetyl cysteine, and ergothioneine, as well as several disulfides, toward different redox states of myoglobin, mainly met-myoglobin (HX-FeIII) and ferrylmyoglobin (HX-FeIV=O), was evaluated by optical spectral analysis, product formation, and thiyl free radical generation. Only dihydrolipoic acid reduced met-myoglobin to oxy-myoglobin, whereas all the other thiols tested did not interact with met-myoglobin. Although the redox transitions involved in the former reduction were expected to yield the dihydrolipoate thiyl radical, the reaction was EPR silent. Conversely, all thiols interacted to different extent with the high oxidation state of myoglobin, i.e. ferrylmyoglobin, via two processes. First, direct electron transfer to heme iron in ferrylmyoglobin (HX-FeIV=O) with formation of met-myoglobin (HX-FeIII) or oxymyoglobin (HX-FeIIO2); the former transition was effected by all thiols except dihydrolipoate, which facilitated the latter, i.e. the formation of the two-electron reduction product of ferrylmyoglobin. Second, nucleophilic addition onto a pyrrole in ferrylmyoglobin with subsequent formation of sulfmyoglobin. The contribution of either direct electron transfer to the heme iron or nucleophilic addition depended on the physicochemical properties of the thiol involved and on the availability of H2O2 to reoxidize met-myoglobin to ferrylmyoglobin. The thiyl radicals of glutathione, cysteine, and N-acetylcysteine were formed during the interaction of the corresponding thiols with ferrylmyoglobin and detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyroline-N-oxide. The intensity of the EPR signal was insensitive to superoxide dismutase and it was decreased, but not suppressed, by catalase. The disulfides of glutathione and cysteine did not react with ferrylmyoglobin, but the disulfide bridge in lipoic acid interacted efficiently with the ferryl species by either reducing directly the heme iron to form met-myoglobin or adding onto a pyrrole ring to form sulfmyoglobin; either process depended on the presence or absence of catalase (to eliminate the excess of H2O2) in the reaction mixture, respectively. The biological significance of the above results is discussed in terms of the occurrence and distribution of high oxidation states of myoglobin, its specific participation in cellular injury, and its potential interaction with biologically important thiols leading to either recovery of myoglobin or generation of nonfunctional forms of the hemoprotein as sulfmyoglobin.     

Novel Mechanism of Hemin Capture by Hbp2, the Hemoglobin-binding Hemophore from Listeria monocytogenes

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2^N2; residues 183–303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2^N2 undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron.     

ABCG2 Transports and Transfers Heme to Albumin through Its Large Extracellular Loop

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K_d values are in the range 0.5–3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K_d of about 3 μm.     

The effect of hemin and of allyl isopropyl acetamide on protein synthesis in rat hepatocytes

Protein synthesis was measured in incubated hepatocytes. While hemin brings about a slight stimulation, allyl isopropyl acetamide (a compound that destroys the heme bound to cytochrome P450) inhibits protein synthesis by a mechanism that appears to result exclusively from depletion of cytoplasmic heme. Indications that in hepatocytes, as in reticulocytes, protein synthesis may be in part regulated by heme at the level of initiation are: i) that inhibition is accompanied by polysome breakdown; ii) that the protein synthesis inhibitor already isolated from rat liver, is hemin reversible iii) that hepatocyte extracts contain a Mr 38,000 phosphoprotein which comigrates with the Mr 38,000 subunit of rabbit initiation factor 2 and iv) that the phosphorylation of both of these subunits is inhibited by hemin.     

Mechanism of horseradish peroxidase-catalyzed conversion of iodine to iodide in the presence of EDTA and H2O2

EDTA not only blocks the horseradish peroxidase (HRP)-catalyzed iodide oxidation to I-3 but also causes an enzymatic conversion of oxidized iodine species to iodide (Banerjee, R. K., De, S. K., Bose, A. K., and Datta, A. G. (1986) J. Biol. Chem. 261, 10592-10597). The EDTA effect on both of these reactions can be withdrawn with a higher concentration of iodide and not with H2O2. Spectral studies indicate a possible interaction of EDTA with HRP as evidenced by the formation of modified compound 1 with H2O2 at 416 nm instead of 412 nm in the absence of EDTA. EDTA causes a hypochromic effect on HRP at 402 nm which undergoes the bathochromic red shift to 416 nm by H2O2. The addition of iodide to the 416 nm complex causes the reappearance of the Soret band of HRP at 402 nm. Among various EDTA analogues tested, N-N-N'-N'-tetramethylethylenediamine (TEMED) is 80% as effective as EDTA in the conversion of I-3 to iodide and produces a spectral shift of HRP similar to EDTA. Interaction of EDTA with HRP is further indicated by the hyperchromic effect of HRP and H2O2 on the absorption of EDTA at 212 nm. The addition of oxidized iodine species produces a new peak at 230 nm due to formation of iodide. EDTA at a higher concentration can effectively displace radioiodide specifically bound to HRP indicating its interaction at the iodide-binding site. The enzyme, after radioiodide displacement with EDTA, shows a characteristic absorption maximum at 416 nm on the addition of H2O2, indicating that EDTA is bound with the enzyme. Both positive and negative circular dichroism spectra of HRP and the HRP.H2O2 complex, characteristic of heme absorption, are altered by EDTA, suggesting an EDTA-induced conformational change at or near the heme region. This is associated with a change of affinity of heme toward H2O2 and azide. It is postulated that EDTA interacts at the iodide-binding site of the HRP inducing a new conformation that blocks iodide oxidation but is suitable to convert iodine to iodide by a redox reaction with H2O2.     

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

The effects of various refolding additives, including metal cofactors, organic co‐solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well‐known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion‐containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca²⁺ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3‐fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea‐containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor‐containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.     

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 microM) at 37 degrees C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (+/-0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (+/-5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78-187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15-36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1-100 microM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to > or = 12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe3+. These contrasting effects of plasma thiols on hemin- and free Fe3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.     

The effects of heme-binding proteins on the peroxidative and catalatic activities of hemin.

The plasma proteins hemopexin (Hx) and albumin (Alb) are known to bind heme with high and medium affinity, respectively. To study how this binding modifies heme catalytic reactivity, the effects of Hx, human serum Alb (HSA), and bovine serum Alb (BSA) on the peroxidase- and catalaselike activities of hemin were investigated. These hemin activities were found to be inhibited by 50 to 60% with either HSA or BSA, and by 80 to 90% with Hx. The heme complexes with Hx or Alb (1:1 = protein:heme) therefore had a much lower reactivity toward H2O2 and Cum-OOH than the nonprotein heme. A kinetic analysis suggested that binding to Hx or Alb inhibited the primary activation of heme by H2O2, the step common for both peroxidase- and catalaselike activities of hemin. It is thought that by complexing heme, the Hx and Alb can prevent the toxic effects of extracellular heme in blood plasma.     

Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins.

Oxidation studies of hemoglobin by Cu(II) indicate that for horse hemoglobin, up to a Cu(II)/heme molar ratio of 0.5, all of the Cu(II) added is used to rapidly oxidize the heme. On the other hand, most of the Cu(II) added to human hemoglobin at low Cu(II)/heme molar ratios is unable to oxidize the heme. Only at Cu(II)/heme molar ratios greater than 0.5 does the amount of oxidation per added Cu(II) approach that of horse hemoglobin. At the same time, binding studies indicate that human hemoglobin has an additional binding site involving one copper for every two hemes, which has a higher copper affinity than the single horse hemoglobin binding site. The Cu(II) oxidation of human hemoglobin is explained utilizing this additional binding site by a mechanism where a transfer of electrons cannot occur between the heme and the Cu(II) bound to the high affinity human binding site. The electron transfer must involve the Cu(II) bound to the lower affinity human hemoglobin binding site, which is similar to the only horse hemoglobin site. The involvement of beta-2 histidine in the binding of this additional copper is indicated by a comparison of the amino acid sequences of various hemoglobins which possess the additional site, with the amino acid sequences of hemoglobins which do not possess the additional site. Zn(II), Hg(II), and N-ethylmaleimide (NEM) are found to decrease the Cu(II) oxidation of hemoglobin. The sulfhydryl reagents, Hg(II) and NEM, produce a very dramatic decrease in the rate of oxidation, which can only be explained by an effect on the rate for the actual transfer of electrons between the Cu(II) and the Fe(II). The effect of Zn(II) is much smaller and can, for the most part, be explained by the increased oxygen affinity, which affects the ligand dissociation process that must precede the electron transfer process.     

Functional binding analysis of human fibrinogen as an iron- and heme-binding protein

Human fibrinogen is a metal ion-binding protein, but its mechanism of binding with iron and heme has not been elucidated in detail. In this study, human fibrinogen was immobilized on CNBr-activated Sepharose 4B beads. The fibrinogen beads bound hemin (iron–protoporphyrin IX: PPIX) as well as iron ion released from ferrous ammonium sulfate (FAS) more efficiently than Sepharose 4B beads alone. Hemin bound to fibrinogen still exhibited pseudo-peroxidase activity. The affinity of fibrinogen binding to hemin, Sn–PPIX, Zn–PPIX and metal-free PPIX followed the order Sn–PPIX < metal-free PPIX < hemin < Zn–PPIX; PPIX bound more non-specifically to control beads. FAS significantly enhanced the binding of hemin to fibrinogen beads. These results suggest that human fibrinogen directly recognizes iron ion, the PPIX ring and metal ions complexed with the PPIX ring, and that the binding of hemin is augmented by iron ions.     

System for the expression of recombinant hemoproteins in Escherichia coli

Expression of recombinant hemoproteins in Escherichia coli is often limited because a vast majority of the protein produced lacks the heme necessary for function. This is compounded by the fact that standard laboratory strains of E. coli have a limited capacity to withdraw heme from the extracellular environment. We are developing a new tool designed to increase the heme content of our proteins of interest by simply supplementing the expression medium with low concentrations of hemin. This hemoprotein expression (HPEX) system is based on plasmids (pHPEX1-pHPEX3) that encode an outermembrane-bound heme receptor (ChuA) from E. coli O157:H7. This heme receptor, and others like it, confers on the host the ability to more effectively internalize exogenous heme. Transformation of a standard laboratory E. coli protein expression strain (BL-21 [DE3]) with the pHPEX plasmid led to the expression of a new protein with the appropriate molecular weight for ChuA. The receptor was functional as demonstrated by the ability of the transformant to grow on iron-deficient media supplemented with hemin, an ability that the unmodified expression strain lacked. Expression of our proteins of interest, catalase-peroxidases, using this system led to a dramatic and parallel increase in heme content and activity. On a per-heme basis, the spectral and kinetic properties of HPEX-derived catalase-peroxidase were the same as those observed for catalase-peroxidases expressed in standard E. coli-based systems. We suggest that the pHPEX plasmids may be a useful addition to other E. coli expression systems and may help address a broad range of problems in hemoprotein structure and function.     

Kinetics of hemoprotein reduction and interprotein heme transfer

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W.T., Liem, H.H., Sutor, R.P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R.F., Gibbs, E.J., Hoeflin, E., Kosar, W.P., Kubera, G., Skowronek, C. A., Wong, N.M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (HmIII., ferriprotoporphyrin IX) was reduced to HmII (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of HmIII to HmII were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K congruent to 10(8)), and with hemopexin (HHx), which binds heme equimolarly (K congruent to 10(13)). The rate of reduction of HmIII to HmII on HSA was first order over several half-lives and linearly dependent on [S2O4(2-)]1/2. At [HSA]0/[HmIII] = 3, the kobsd was (5 X 10(-3) + 0.75[S2O4(2-)]1/2, and with [HSA]/[HmIII] approximately 25, the kobsd was (2 X 10(-3)) + 0.25[S2O4(2-)]1/2. The reduction of HmIII to HmII on human hemopexin (HHx) is much more rapid with kobsd = (2.5 X 10(3))[S2O4(2-)]1/2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.