Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. M?ssbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.     

Inhibition of zinc absorption by iron depends on their ratio

Previous studies upon zinc-iron interactions gave conflicting results that could come from differences in protocol design or in trace element status of subjects. The present work assessed the influence of zinc : iron ratio and iron deficiency upon zinc absorption. The digestive absorption of zinc sulphate (100 mol Zn/l) in presence of iron gluconate was studied in perfused jejunal loops (n = 6/group) of normal rats (range 0–1000 mol Fe/l) and iron deficient rats (200–750 mol Fe/l). In normal rats no significant iron inhibition on zinc absorption occurred at Fe:Zn ratio below 2:1. At higher ratios zinc uptake and net absorption decreased significantly (p<0.05). Between 2:1 and 5:1 a dose dependent inhibition of zinc absorption occurred and reached a plateau beyond this ratio. In iron deficient animals no changes in zinc uptake, mucosal retention and absorption compared to normal animals occurred at ratio 2:1. At higher ratios differences were observed at every zinc absorption step except for mucosal retention at 7.5:1 ratio.

Iron-zinc interactions depend on their ratio and on previous trace elements status of subjects. Due to the wide and unknown variations that were likely to occur between the subjects of previous human and experimental studies, these results could explain some of the discrepancies between their results.     

Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen

The anticancer drug adriamycin binds iron and these complexes cycle to reduce molecular oxygen (Zweier, J. L. (1984) J. Biol. Chem. 259, 6056-6058). Optical absorption, EPR, and M?ssbauer spectroscopic data are correlated with polarographic O2 consumption and chemical Fe2+ extraction measurements in order to characterize each step in this cycle. Fe3+ binds to adriamycin at physiologic pH forming a complex with an optical absorbance maximum at 600 nm. EPR signals at g = 4.2 and g = 2.01, and a doublet M?ssbauer spectrum with isomer shift delta = 0.57 mm/s and quadrupole splitting delta EQ = 0.74 mm/s are observed indicating that the Fe3+ bound to adriamycin is high spin S = 5/2. Under anaerobic conditions the absorbance maximum at 600 nm decreases with an exponential decay constant = 0.77 h-1, and the EPR and M?ssbauer spectra of Fe3+-adriamycin similarly decrease as the Fe3+ is reduced to EPR silent Fe2+. The Fe2+-adriamycin complex which is formed exhibits a M?ssbauer spectrum with delta = 1.18 mm/s and delta EQ = 1.82 mm/s indicative of high spin Fe2+. As the EPR spectra of Fe3+-adriamycin decrease on reduction of the Fe3+ to Fe2+ a signal of the oxidized adriamycin free radical appears at g = 2.004 with line width of 8 G. On exposure to O2 the absorption maximum at 600 nm, the Fe3+ EPR, and the Fe3+ M?ssbauer spectra all return. Polarographic measurements demonstrate that O2 is consumed and that H2O2 is formed. Addition of high affinity Fe2+ chelators block O2 consumption indicating that Fe2+ formation is essential for O2 reduction. This cycle of iron-mediated O2 reduction can explain the formation of the reactive reduced oxygen and adriamycin radicals which are thought to mediate the biological activity of adriamycin.     

The interactions of haem with ligandin and aminoazo-dye-binding protein A.

1. The interactions of ferriprotoporphyrin IX with ligandin and aminoazo-dye-binding protein A result in absorption spectra in the Soret region characteristic of the ligand in its monomeric state. 2. Both proteins are able to bind ferrous as well as ferric haem. 3. Ferriprotoporphyrin IX is bound at a single site on both proteins. At pH7.0, I 0.16M, difference-spectrophotometric measurements gave association constants of 10(7) and 4 X 10(6) LITRE/MOL FOR LIGANDIN AND PROTEin A respectively. Under the same conditions fluorescence-quenching experiments gave an association constant of 2 X 10(7) litre/mol for ligandin. 4. Bilirubin, bromosulphophthalein and oesterone sulphate each compete with haem for binding by the two proteins. 5. Ferriprotoporphyrin IX bound to both ligandin and protein A is able to form co-ordination complexes with CN-, but not, to any measurable extent, with either N3- or F-. From these results it is suggested that binding by the two proteins may not involve the haem iron atom. 6. Both haem-protein complexes give rise to measurable extrinsic Cotton effects in the Soret region. 7. The formation and properties of the ligandin- and protein A-haem complexes are compared with those of haem-albumin, haemoglobin, myoglobin and other haemoproteins.     

Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.     

EPR and M?ssbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K M?ssbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The M?ssbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and M?ssbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the M?ssbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.     

A rubredoxin-like mononuclear FeS4 derivative of adrenal iron-sulfur protein (adrenodoxin)

A rubredoxin-like mononuclear iron-sulfur derivative of adrenodoxin was prepared from the apoprotein and FeCl₃ in the presence of dithiothreitol. The mononuclear compound displayed optical absorption maxima at 276, 350, and 500 nm, and exhibited electron paramagnetic resonance absorption at g = 4.27 with a shoulder at g = 4.28, which can be ascribed to high spin ferric ion. From p-chloromercuriphenyl sulfonate titration experiments the iron atom appears to contain approximately one g atom of iron per mole of protein. This rubredoxin-like derivative was very unstable at 22° (the half-life was approximately 10 minutes), whereas the native 2 Fe2S1 protein is known to be quite stable. This instability is believed to be intrinsic to the polypeptide sequence of adrenodoxin.     

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).     

The role of iron in the activation of mannonic and altronic acid hydratases, two Fe-requiring hydro-lyases

D-Altronate hydratase and D-mannonate hydratase belong to a class of Fe2+-requiring enzymes, but the function of iron in these enzymes is largely unknown. Methods are described for the convenient preparation of both these hydratases from Escherichia coli and studies related to metal activation are presented. The enzymes are inactive in the absence of a bivalent metal and a reducing agent such as dithiothreitol. Fe2+ at low concentrations activates the enzymes efficiently, but inhibits them over 2 mM. Furthermore Mn2+ is also capable of activating aldonic acid hydratases and appears to be a constituent of the enzyme active center. A marked synergistic activation is observed in the presence of both ions, raising the possibility that the enzyme has two binding sites for ions. Upon activation, the two aldonic acid hydratases incorporate a single Fe atom and contain no Fe-S core, in contrast to other characterized Fe-hydratases, such as aconitase or maleic acid hydratase. The incorporated iron is losely bound (with Kd about 4.5 mM and 20 mM for mannonate and altronate hydratase, respectively) and can be readily removed with EDTA. The enzymes exhibit no requirement for sulphide ions and are insensitive to thiol reagents. A first-order inhibition is observed with iron chelators and can be removed by competition with excess metal ions. No change in the absorption spectra is observed upon oxidation-reduction or activation with metals. The activated enzymes exhibit no electron paramagnetic (EPR) spectrum under anaerobic conditions; in the presence of oxygen, an intense EPR spectrum develops in Fe2+-activated samples with signal at g = 1.98, which upon reaction of the enzyme with the substrate moves into a species with signals at g = 4.15 and g = 9.07, with EPR parameters very similar to those of oxidized rubredoxins.     

Intestinal iron absorption in chickens

Intestinal iron absorption in chickens was studied in vivo, using an intestinal perfusion technique in closed circuit. The results obtained show that iron absorption, at 30 min intervals, is a linear function of test solution iron concentrations of up to 776 μg Fe/20 mL. At higher concentrations, iron saturation occurs. The mucosal epithelial cells seem to be less a limiting factor than in rats. However, in chickens, the binding capacity of plasma might play an important role in the regulation of iron absorption. Iron absorption versus time was analyzed in 15, 30, 60, and 120 min periods for the iron concentration of 14 μg Fe/20 mL. Intestinal iron absorption showed a linear relationship between these two parameters. A period of perfusion of either 30 or 60 min by a solution of 14 μg Fe/20 mL appears suitable since no interference by a saturation process can then occur.     

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses.ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.     

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

Dengue haemorrhagic fever and dengue shock syndrome: are they tumour necrosis factor-mediated disorders?

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.     

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.     

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.     

Oxygen equilibrium study and light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.     

Fe-S centers in lactyl-CoA dehydratase

Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/- 0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/- 0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic interactions of CO with a3Fe and CuB in cytochrome c oxidase in beef heart mitochondria studied by Fourier transform infrared spectroscopy at low temperatures

Carbon monoxide bound to cytochrome c oxidase has been observed by Fourier transform infrared spectroscopy between 10 K and 280 K in the dark and during and after continuous photolysis. CO bound to a3Fe absorbs near 1963 cm-1, with minor bands at lower frequencies. Photolysis at low temperatures transfers CO to CuB, with the major component near 2062 cm-1 and a minor one near 2043 cm-1. Vibrational absorptions are assigned by comparison with heme and copper carbonyls, by frequency dependence of all bands on the isotopic mass of CO, and by similar behavior of major and minor components with photolysis and relaxation kinetics as a function of temperature. Reformation of a3FeCO after photolysis is an apparent first order process below 210 K with a distribution of rate constants. The kinetics are well described by a power law. Arrhenius behavior is followed between 140 K and 180 K to yield a peak activation enthalpy of 40.3 kJ/mol and a distribution in g(H) = 2.56 kJ/mol (full width at half-maximum). The major component of a3FeCO shows a very narrow CO absorption band (full width at half-maximum = 2.4 cm-1), while that of CuBCO shows a broader CO absorption (full width at half-maximum = 6 cm-1). These data indicate that in the reduced carbon monoxide complex, a3FeCO is in highly ordered nonpolar surroundings sufficiently separated from CuB that it is not perturbed by motion of the latter, while CuBCO is in less ordered, more flexible surroundings.     

Modeling low-pH hemoproteins

A tetracoordinate ferrous heme (iron-porphyrin) has been proposed as an intermediate at low pH (less than 3.0) for respiratory hemoproteins, peroxidases, and model heme complexes. This intermediate is believed to arise via protonation of the N(epsilon) atom of the proximal histidine and consequent cleavage of the Fe-N(epsilon) bond. To establish a spectral signature for the proposed low-pH tetracoordinate species, we have obtained Soret excitation resonance Raman spectra on samples of crystallographically defined, tetracoordinate iron(II)-octaethylporphyrin (Fe.OEP; S = 1). The high-frequency (greater than or equal to 900 cm-1) resonance Raman spectral features of Fe.OEP are clearly distinct from those of high-spin pentacoordinate or low-spin hexacoordinate ferrous hemes. Rather, they are at frequencies more typically observed for low-spin hexacoordinate ferric porphyrins. Comparative spectral analysis of tetracoordinate Fe.OEP and other proposed tetracoordinate ferrous hemes (e.g. iron(II)-protoporphyrin IX) demonstrates little or no macrocycle effect on the resonance Raman frequencies above 900 cm-1. This work thus serves to provide a testable spectral signature by which the existence of the proposed tetracoordinate biological intermediate may be verified and by which its functional significance may be tested.