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We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm-1, with relative intensities that are pH sensitive. The 534 cm-1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogen-bonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm-1 is assigned as being associated with the 534 cm-1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm-1, respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm-1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.

The antitumor drug, bleomycin, interacts with either Fe(II) and O 2 or Fe(III) and H2O2 to form an activated complex which attacks DNA. Under aerobic conditions, both reactions yield similar quantities of free bases and products consisting of base plus deoxyribose carbon atoms 1 to 3. Under anaerobic conditions, activated bleomycin releases only free base. The yield of free base is the same under aerobic or anaerobic conditions, provided DNA is furnished in excess. When the DNA concentration is limiting, more base is released under anaerobic than under aerobic conditions. Drug self-destruction proceeds as quickly and completely in the presence or absence of O2.

Activated bleomycin is shown for the first time to cleave tRNA in a specific and dose-dependent manner. Adenine and uracil are released in the reaction. Bleomycin and Fe(III)-bleomycin bind to yeast tRNAPhe) in analogy with the known behavior of the drug with B-DNA.

The binding of the iron complex of the antineoplastic glycopeptide bleomycin A2 (Fe-BLM) to calf thymus DNA and the self-complementary oligonucleotides d(CGCGCG) and d(ATATAT) has been studied using optical, EPR, and resonance Raman spectroscopies. An increase in the intensity of the bands at 365 and 384 nm is observed in the optical spectrum of Fe(III)-BLM when the drug binds to either oligonucleotide. However, in the presence of phosphate, this increase is observed only with d(CGCGCG) and not with d(ATATAT). In addition, the gmax feature in the EPR spectrum of low spin Fe(III)-BLM is narrowed in a way suggesting a reduction of possible conformers that the drug can achieve when it is bound to d(CGCGCG) or to calf thymus DNA but not when bound to d(ATATAT). When Fe(III)-BLM is bound to d(CGCGCG), changes in the resonance Raman spectrum of the metal drug complex suggest conformational changes in three of the ligands to iron: the beta-hydroxyhistidyl amide, the pyrimidine, and the axial hydroxide. In addition, the Fe-OH band undergoes narrowing, again consistent, with the reduction of conformers of the drug. No such resonance Raman changes are observed upon binding to d(ATATAT). The changes in the pyrimidine modes upon binding d(CGCGCG) to the drug are consistent with a recently proposed model (Wu, W., Vanderwall, D. E., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1996) J. Am. Chem. Soc. 118, 1281-1294) of DNA recognition by activated bleomycin, HOO-Fe(III)-BLM, in which the pyrimidine moiety of the drug is important for the preferential cleavage of 5'-GpPy-3' sequences.

Activated bleomycin appears to have two more oxidizing equivalents than the Fe(III).bleomycin to which it spontaneously decays. Activated bleomycin reacts with NADH and thio-NADH, two-electron reductants, and with KI, a one-electron reductant, to yield Fe(III).bleomycin. The observed stoichiometries were 0.85 +/- 0.07 eq of thio-NADH oxidized or 1.5 +/- 0.25 eq of KI oxidized per mole of activated bleomycin. None of these reactions requires the presence of a redox mediator, as does the reduction of Fe(III).bleomycin by NADH or thio-NADH. The oxidations of both pyridine nucleotide coenzymes and of KI are inhibited by DNA, the usual bleomycin target.

Hemoglobin A and hemoglobin Kansas can exist in alternative forms in which the affinity of heme iron for NO is mediated either by pH or the presence of allosteric modifiers. In this study, we show that the superhyperfine (SHF) pattern observed in the EPR which arises from the heme iron‐NO complex can be correlated with a change in the affinity of the ligated tetrameric protein for NO. When a combination three‐line and nine‐line SHF pattern is observed, the nitrosyl hemoprotein is in a low affinity form while a nine‐line pattern alone is an expression of high affinity. A theory is presented relating SHF patterns to a molecular orbital picture of the nitrosyl heme complex. It is suggested that the change of SHF pattern correlated with an alteration of the affinity state of ligated hemoglobin is related to a structural change of the proximal imidazole ligand to the heme.

Cellular copper metabolism and the mechanism of resistance to copper toxicity were investigated using a wild type hepatoma cell line (HAC) and a copper-resistant cell line (HAC600) that accumulates copper and has a highly elevated level of metallothionein (MT). Of the enzymes involved in reactive oxygen metabolism, only glutathionine peroxidase was elevated (3–4-fold) in resistant cells, suggestive of an increase in the cellular flux of hydrogen peroxide.

The action of iron-bleomycin and O2 in cleaving DNA has been resolved into two kinetic events following the initial attack on DNA by the kinetically competent drug species, "activated bleomycin." At 4 degrees C, DNA strand scission, monitored both viscometrically and fluorimetrically (t1/2 = 2.5-5 min), precedes the release from DNA of nucleic base propenals, which is half complete in about 40 min. Therefore, a moderately stable intermediate consisting of cleaved DNA bearing a base propenal precursor is formed. The release of tritium from deoxyribose carbon-2 occurs at the time of DNA scission, which is consistent with the base propenal precursor retaining the deoxyribose-3'-phosphate bond. Specific mechanistic proposals are discussed.

The properties of sulfhemoglobin (sulfHb) were investigated using disc gel isoelectric focusing and optical spectrophotometry. Laboratory-prepared samples, which contained a high yield of sulfHb (70-85%), and a patient-derived sample, which contained a low yield (12%), contain a tetrameric population that reflects a random distribution of modified (sulfurated) subunits. Hybrid tetramers, i.e. those containing sulfurated and unmodified subunits, were resolved from fully sulfurated and unmodified hemoglobin upon electrofocusing of ferric or ferrous sulHb samples. The electrophoretic differences between ferric sulfHb and metHb arise from a difference in the pK for the met to hydroxymet conversion of sulfurated subunits (Carrico, R., Peisach, J., and Alben, J. O. (1978) J. Biol. Chem. 253, 2386-2391). Both partially and fully sulfurated tetramers, when in the deoxy form and when in the fully ligated CO form, co-focus with their unmodified deoxy-Hb and Hb CO counterparts. Thus, these sulfurated tetramers exhibit normal ligand-dependent ionization of Bohr protons. In air-equilibrated samples, hybrid tetramers are partially ligated as a result of the reduced O2 affinity of the sulfurated subunits. These partially ligated tetramers exhibit a pI intermediate between oxy-Hb and deoxy-Hb due to the fractional ionization of Bohr protons. The partially sulfurated, partially ligated tetramers as well as deoxy-sulfHb tetramers were found to bind 2,3-diphosphoglycerate in disc gels. Despite the preservation of these heterotropic effects, which are confirmed in CO binding studies in solution, fully sulfurated tetramers recovered using preparative isoelectric focusing exhibit little or no cooperativity.

Fe(III)-bleomycin associates strongly with rat liver nuclei and binds to nuclear DNA. Metal-free and Cu(II)-bleomycin, however, do not bind to nuclei. The treatment of nuclei with activated iron-bleomycin results in nucleic base and base propenal release from the DNA, and also gives membrane peroxidation. Isolation and quantitation of the base propenals and free bases released subsequent to activated bleomycin treatment reveal an alteration in the stoichiometry of these products compared to those released from purified DNA. With nuclei, significantly less propenal is formed, although the yield of free base is equivalent to that from purified DNA. The membrane peroxidation products from nuclei are the same as those obtained from microsomal membranes treated with activated bleomycin. Superoxide dismutase inhibits the membrane peroxidation but has no effect on the DNA breakage reactions. The results implicate a role for iron in mediating the in vivoaction of bleomycin and also reveal a potentially toxic effect, membrane peroxidation, separate from DNA damage.

From a study of the magnetic field dependence of the linear electric field effect (LEFE) in EPR spectroscopy, we demonstrate that iron-sulfur cluster 3 in air-oxidized, beef heart succinate-ubiquinone oxidoreductase (Complex II) is a 3-iron cluster. This suggests that cluster 3 may arise by oxidative degradation from a 4-iron cluster originally present in the enzyme and may be reconverted back into a 4-iron cluster under reducing conditions. Linear electric field effect studies of succinate-reduced Complex II are in accord with the view that cluster 1 is a 2-iron cluster.

A series of four cell lines resistant to the toxic effect of copper were developed from Morris rat hepatoma cells by gradually increasing the concentration of copper in the growth medium. The EC50, that concentration of copper that kills and/or inhibits the growth of 50% of the cells after 72 h, increased 4-fold over that for wild type cells in the most resistant cell line. These cells were also resistant to zinc, cadmium, and mercury toxicity, but not to nickel or cobalt. The amount of copper in the soluble protein pool of the resistant cells increased proportionally with the concentration of copper in the medium in which they were maintained. Associated with copper accumulation was the production of an 18-kDa cysteine-rich protein which complexes a significant amount of the metal. It is suggested that resistance to copper toxicity is due to sequestration of the metal by this protein. When resistant cells were removed from the copper-enriched environment, cellular copper levels rapidly fell to that observed for wild type cells, but no reduction in either the EC50 or the level of the cysteine-rich protein was noted. This suggests that a permanent change responsible for copper resistance had occurred which is maintained in the absence of the metal.

Porphobilinogen oxygenase oxidizes porphobilinogen to 2-hydroxy-5-oxo-porphobilinogen. This enzyme isolated from wheat germ has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions. The molecular weight of the enzyme formed from two identical (or very similar) polypeptide chains is 70,000. It has a pI of 9.0 indicating its cationic nature. The pure enzyme contains 1 mol of high-spin heme and 2 mol of non-heme iron. It requires both of these as well as molecular O2 and a reducing agent for catalytic activity. Although the enzyme has many characteristics of a peroxidase, hydrogen peroxide cannot substitute for oxygen and dithionite for catalysis. The catalytic reaction is not affected by catalase, superoxide dismutase, or by hydroxyl radical scavengers. A comparison between porphobilinogen oxygenase and a commercial preparation of horseradish peroxidase was made. The latter also catalyzes aerobic porphobilinogen oxidation, with dithionite as electron donor. Here the oxidation of porphobilinogen is inhibited by superoxide dismutase and was not affected by catalase.

Whereas phagocytic cells from normal individuals have the capacity to kill ingested bacteria and parasites, those from patients with several uncommon genetic deficiency diseases are known to be defective in bactericidal activity. Studies on neutrophils of these patients have revealed fundamental defects in their ability to reduce molecular oxygen and metabolize it to superoxide anion, hydrogen peroxide, and oxygen radicals. In the present experiments, we describe a clone of a continuous murine macrophage-like cell line, J774.16, that, upon appropriate stimulation, activates the hexose monophosphate shunt, and produces superoxide anion and hydrogen peroxide. With nitroblue tetrazolium to select against cells capable of being stimulated by phorbol myristate acetate to reduce the dye to polymer--formazan--which is toxic fot cells, we have selected for variants that are defective in oxygen metabolism. Four of these subclones have been characterized and found to be lacking in the ability (a) to generate superoxide anion, as measured by cytochrome c reduction; (b) to produce hydrogen peroxide, as measured by the ability to form complex I with cytochrome c peroxidase; and (c) to be stimulated to oxidize glucose via the hexose monophosphate shunt. These variants appear to represent a useful model for studying the molecular basis for macrophage cytocidal activity.

The inhibition of Fe(II)-bleomycin activation, by a large excess of DNA, is overcome by rat liver microsomes in the presence of NADPH. This release of inhibition, as indicated by increased yields of base propenal from DNA scission, is enhanced by menadione, is inhibited by superoxide dismutase, and is therefore dependent on superoxide anion. Microsomal activation of Fe(II)-bleomycin doubles the stoichiometry of base propenal yield compared to that obtained upon self-activation of the drug; 0.5 mol of base propenal is formed and 0.5 mol of NADPH is oxidized per mol of Fe(II)-bleomycin. In the presence of a large excess of DNA, Cu(II)-bleomycin is not reduced and Fe(III)-bleomycin is neither reduced nor activated by microsomes in cases where activation of Fe(II)-bleomycin is maximal. We suggest that in vivo, electron transport enzymes at or near the nucleus can stimulate the activation of Fe(II)-bleomycin under conditions where self-activation does not readily occur.

Using Mössbauer spectroscopy, we have examined iron-bleomycin in various oxidation states and in complexes with dioxygen or carbon monoxide. Ferrous bleomycin is a high spin ferrous complex. Addition of O2 converts it into an EPR-silent oxygenated complex. Mössbauer studies in strong applied magnetic fields show that oxygenated bleomycin is diamagnetic. At 4.2 K, the quadrupole splitting delta EQ = -2.96 mm/s and the isomer shift delta = 0.16 mm/s suggest that its electronic structure is best described as low spin ferric iron bound to superoxide anion. A single electron reduction yields activated bleomycin, an EPR-active form that still retains oxygen and which is kinetically competent to initiate DNA cleavage. We have produced this complex by exposing ferrous bleomycin to O2 or by reacting ferric bleomycin with H2O2. The Mössbauer spectra give convincing evidence that the iron of activated bleomycin is low spin ferric. The decay of activated bleomycin yields low spin ferric bleomycin, a complex with Mössbauer parameters nearly identical with those reported for ferric cytochrome P-450. Although iron bleomycin does not have a polyaromatic structure like heme, many features of its electronic structure at the iron are very similar to those produced by the sulfur-coordinated heme iron of ferric cytochrome P-450, a protein that catalyzes a similar oxygen-dependent reaction.

Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate.

All heme proteins containing mononuclear high spin ferric heme, when examined at low temperatures (near 1°K), exhibit X-band electron paramagnetic resonance (EPR) absorptions extending from near g= 6 to g= 2 which arise from transitions of the lowest Kramers doublet. Meaningful absolute quantitation of high spin ferric EPR spectra cannot be made other than from experiments at very low temperature or from a knowledge of the zero field splitting.

Ferrous sulfmyoglobin was prepared from the higher oxidation state of myoglobin (Mbiv) by reaction at pH 8 with a 1.5-fold molar excess of (NH4)2S. The purity (>90%) was greater than any previously reported.

Oxyhemoglobin either spontaneously or under the influence of external oxidants is converted to the high spin ferric protein (acid methemoglobin) which can be studied both optically and with electron paramagnetic resonance (EPR). The rate of ferricyanide oxidation is comparable for oxyhemoglobin A, oxyhemoglobin H (β4), and oxy α chains. p-Mercuribenzoate (PMB) derivatives of oxy α and oxy β chains react faster with this reagent than do the nonderivatized proteins.

The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putidawere observed at temperatures from 4.2 to 80°K. As isolated, P-450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g= 2.45, 2.26, 1.915). We also detected a minor signal typical of high spin ferric heme (g= 8, 4, 1.8) equivalent to less than 7% of the heme at temperatures below 20°K. On titration with the substrate, (+)-camphor, the low spin signal decreased and the high spin signal increased, maximally representing about 60% of the heme. For reasons not thus far understood, 40% of the heme is not converted to high spin by either (+) or (-)-camphor. The high spin signal has a rhombic character which is stronger than any previously observed with a heme compound (E= 0.33 cm-1; D= 3.8 cm-1; E/D= 0.087).We conclude that P-450camas isolated is equal to or more than 95% in a low spin form probably having sulfur as one of the axial ligands. The binding of substrate displaces this ligand sufficiently to allow for conversion from a low to a high spin form.

The ferrous form of sulfmyoglobin was isolated as the first stable product of the reaction of a single mole of inorganic sulfide with 1 mole of the higher oxidation state derivative of myoglobin, Mbiv. Using radioactive sulfide, it was demonstrated that a single g atom of sulfur is incorporated per mole of myoglobin to form sulfmyoglobin.