Spin Form Research Articles

Solubilisation of ferricytochrome C in methanol using a crown ether: absorption, circular dichroism and epr spectral properties

Ferricytochrome c is normally insoluble in methanol, but its solution is facilitated by complexation with 18-crown-6. Absorption, circular dichroism and EPR spectroscopy indicate that the solubilised protein in MeOH exists in at least three conformational states, all different from the native state in neutral aqueous solution. In two states the haem iron (III) is low spin and in one state it is high spin, but it seems likely that all three forms are globular. The proportion in the high spin form increases at increasing crown ether concentration and on ageing the protein solution. The protein appears to return to its native conformation when it is restored to an aqueous environment.

Interaction of Trichloroethane Isomers with Cytochrome P-450 in the Perfused Rat Liver

Interaction of Trichloroethane Isomers with Cytochrome P-450 in the Perfused Rat Liver. TAKANO, T., MIYAZAKI, Y., and MOTOHASHI, Y. (1985). Fundam. Appl. Toxicol. 5, 353–360. The real-time interactions of 1,1,1-trichloroethane (TCE) and 1,1,2-TCE with cytochrome P-450 were observed using in vivo optical methods to measure the spectral changes of cytochrome P-450 and the reduction-oxidation transition of pyridine nucleotides in the perfused liver of rats treated with phenobarbital. Changes in oxygen consumption and TCE uptake were also measured. The spectral changes of cytochrome P-450 indicated that both TCE isomers bound to low spin (substrate free) ferric cytochrome P-450 and formed a high spin (substrate complexed) form. However, 1,1,1-TCE bound more tightly to cytochrome P450 and seemed to be only slowly metabolized compared to 1,1,2-TCE. The stoichiometry of the change in oxygen consumption rate to the change in 1,1,1-TCE uptake rate ranged between 5/1, and 9/1, whereas that of 1,1,2-TCE was 1.4 to 2.0. Decreases in reduced pyridine nucleotides associated with TCE administration were significantly larger with 1,1,1-TCE than with 1,1,2-TCE. The inhibitory effect of 1,1,1-TCE on hexobarbital metabolism in the perfused liver was greater than that of 1,1,2-TCE. Considering our previous data indicating that TCE did not stimulate mitochondrial respiration, it is postulated that the far higher amount of oxygen consumption associated with the binding of 1,1,1-TCE to cytochrome P450 than the amount which was necessary to mixed-function oxidation of this compound was due to an uncoupling effect of 1,1,1 -TCE on the mixed-function oxidase system.

Activation and Inactivation of a Variety of Mutagenic Compounds by the Reconstituted System Containing Highly Purified Preparations of Cytochrome P-450 from Rat Liver

Activation and Inactivation of a Variety of Mutagenic Compounds by the Reconstituted System Containing Highly Purified Preparations of Cytochrome P-450 from Rat Liver. KAWANO, S., KAMATAKI, T., MAEDA, K., KATO, R., NAKAO, T., AND MIZOGUCHI, I. (1985). Fundam. Appl. Toxicol. 5, 487–498. Six cytochrome P-450 preparations from phenobarbital (PB)-treated rats and two preparations from β-naphthoflavone (BNF)-treated rats were purified. Using Salmonella typhimurium TA98 the ability of these cytochrome P-450 preparations to mutagenically activate and inactivate a variety of carcinogens was examined. High- and low- spin forms of cytochrome P-448 isolated from BNF-treated rats (BNF-IIa and IId) activated various carcinogens. Both forms activated 2-aminofluorene, benzo[a]pyrene, and dibenz[a,c]anthracene. However, o-aminoazotoluene and 2-nitrofluorene were activated only by the low-spin form, and aflatoxin B1 only by the high-spin form. In contrast, only limited carcinogens were activated by some preparations from PB-treated rats. 2-Aminofluorene was activated by four PB-inducible preparations (PB-Ia, Ic, Id, and IIa), but only moderately. Unexpectedly, however, the most prominent activation of benzo[a]pyrene was observed with one preparation (PB-Id) from PB-treated rats. Direct mutagens to the S. typhimurium, 4-NQO and AF-2, were markedly inactivated by NADPH-cytochrome c (P-450) reductase without cytochrome P-450, One PB-inducible form (PB-Ic) inactivted 2-nitrofluorene, and the high- spin form of P-448 (BNF-IIa) inactivated AF-2

Spin state control of cytochrome P-450 reduction and catalytic activity in a reconstituted P-450 LM2 system as induced by a series of benzphetamine analogues

Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.

Heme-linked spectral changes of the protein moiety of hemoproteins in the near ultraviolet region.

Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computer-controlled spectrophotometry. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm. Various difference spectra of hemoglobin in the near ultraviolet region were also measured. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.

Induction of the polysubstrate monooxygenase system of the native budworm Heliothis punctiger (Wallengren) (Lepidoptera: Noctuidae)

The effects of dietary inducers on properties of the polysubstrate monooxygenase system were investigated in the midgut, fat body and integument of Heliothis punctiger larvae. Diets tested were an artificial diet (AD), AD + α-pinene, AD + phenobarbital, and the whole plant diets of tobacco and lucerne. The tobacco and α-pinene diets induced forms of P-450 in midguts with a comparatively low catalytic capacity for aldrin and a high proportion of hemoprotein in the high spin form. In contrast, lucerne and phenobarbital promoted P-450 species with a high affinity for aldrin and hemoprotein predominantly in the low spin state. P-450 specific activity and n- octylamine binding studies revealed that in the fat body phenobarbital pretreatment induced different forms of cytochrome P-450 compared to that predominating in larvae fed on diets high in secondary plant substances. All diets promoted predominantly low spin state P-450 in both the fat body and integument.

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450.

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase.

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.

Cytochrome P-450 in culture forms of Trypanosoma cruzi

1. Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40–60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. 2. Epimastigotes show induced peptides of mol. wt 56,000, 52,500, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. 3. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. 4. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. 5. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. 6. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.

A high spin form of cytochrome P-448 highly purified from PCB-treated rats—II: Characteristic requirement of cytochrome b5 for maximum activity

A high spin form of cytochrome P-448 (PCB P-448-H), highly purified from microsomes of PCB-treated rats, catalyzed oxidations of several compounds and required cytochrome b5 for its full activities in all oxidations examined. PCB P-448-H catalyzed the hydroxylation of aniline and O-dealkylations of p-alkoxy derivatives of aniline and nitrobenzene and 7-alkoxy derivatives of coumarin. Among the activities measured, hydroxylation of aniline and O-dealkylation of p-alkoxy derivatives of aniline were catalyzed by PCB P-448-H more efficiently than by PCB P-448-L, which was a low spin form of cytochrome P-448 purified from liver microsomes of PCB-treated rats. In all reactions, PCB P-448-H required cytochrome b5 for maximum activity. Slight requirements were also seen with PCB P-448-L but varied equivocally depending on the substrates. Cytochrome b5 showed its maximum effects on p-propoxyaniline O-depropylation activity at a molar ratio of cytochrome b5 to PCB P-448-H of 1:2. The enhancement by cytochrome b5 was more pronounced when lower concentrations of either the substrate or NADPH-cytochrome P-450 reductase were added to the reconstituted system. Based on these results, we confirm that PCB P-448-H is a unique form of cytochrome P-448 with respect to the requirements for cytochrome b5 and is a good probe to study the mechanisms involved in the enhancement of drug oxidations by cytochrome b5.

Induced neutron magnetic form factor of LaSn 3

Polarized neutron scattering techniques have been used to study the spatial distribution of the magnetization induced in a single crystal of LaSn 3 by a magnetic field of 42.5 kG at 100 K. We find that the magnetic form factor decreases very rapidly with increasing scattering angle, and bears no resemblance to the spin or orbital free-atom magnetic form factor. The experimental results are in reasonable agreement with band theoretical calculations of the spin magnetic form factor of LaSn 3. We conclude that (a) the spin part is the dominant contribution to the bulk susceptibility of LaSn 3 and (b) there is a substantial amount of Sn-5p electronic character in the wavefunctions near the Fermi level.

Magnetic circular dichroism and spin equilibrium of methemoglobin and its subunits

The intensity of the Soret magnetic circular dichroism (MCD) spectra of various complexes of methemoglobin subunits (α + and β +) as well as methemoglobin (metHb A) was correlated well with the spin states of ferric heme. Upon the subunit association, spin state transition toward higher spin was observed only in high spin derivatives and the changes in spin state were due to mainly those of β + chains. The effect of an allostric effector, inositol hexaphosphate (IHP), on the MCD spectra of metHb A derivatives was observed much significantly for high spin forms than low spin ones.

A globular high spin form of ferricytochrome c.

Acidification of a salt-free solution of native low spin globular horse heart ferricytochrome c with HCl causes a single cooperative transition centered at pH 2.5 at 23 degrees C resulting in the formation of denatured high spin ferricytochrome c. By contrast, acidification with HCIO4 uncouples denaturation from the spin-state transition, resulting in the formation of a globular high spin form of ferricytochrome c at pH 0.0 exhibiting only modest conformational differences from native cytochrome c as judged by far ultraviolet circular dichoroic, tryptophan fluorescence, and reduced viscosity measurements. This uncoupling is consistent with the preferential binding of two perchlorate anions to the globular form(s) cytochrome c. The acid spin-state transition of the perchlorate complex is biphasic, having midpoint values at pH 0.7 and 3.6 involving 1.0 and 1.6 protons, respectively, when measured in the presence of 1 M perchlorate.

N-Hydroxylation of Carcinogenic and Mutagenic Aromatic Amines

N-Hydroxylation and mutagenic activation of heterocyclic aromatic amines from protein pyrolysis products were studied in rat liver microsomes and nuclei, rat hepatocytes and various species of purified cytochrome P-450. These mutagenic amines include Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), Glu-P-1 (2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole), Glu-P-2 (2-amino-dipyrido[1,2-a:3',2'-d]imidazole) and IQ (2-amino-3-methyl-3H-imidazo-[4,5-f]quinoline). The number of revertants of Salmonella typhimurium TA 98 was always correlated to the amount of each of the N-hydroxylated metabolites in various experimental conditions. The N-hydroxylated amines covalently bound to DNA directly or after being acylated with amino acids by amino-acyl-tRNA synthetase. Various species of cytochrome P-450 preparations showed markedly different activity in N-hydroxylation and mutagenic activation of Trp-P-2, Glu-P-1 and IQ. A high spin form of cytochrome P-450, isolated from the liver of PCB-treated rats, showed very high activity in N-hydroxylation of Trp-P-2, Glu-P-1 and 2-aminofluorene, although its activity was very low in benzo(a)pyrene hydroxylation. The present results indicate that different species of cytochrome P-450 are involved in the N-hydroxylation and mutagenic activation of aromatic amines.

N-hydroxylation of carcinogenic and mutagenic aromatic amines.

N-Hydroxylation and mutagenic activation of heterocyclic aromatic amines from protein pyrolysis products were studied in rat liver microsomes and nuclei, rat hepatocytes and various species of purified cytochrome P-450. These mutagenic amines include Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), Glu-P-1 (2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole), Glu-P-2 (2-amino-dipyrido[1,2-a:3',2'-d]imidazole) and IQ (2-amino-3-methyl-3H-imidazo-[4,5-f]quinoline). The number of revertants of Salmonella typhimurium TA 98 was always correlated to the amount of each of the N-hydroxylated metabolites in various experimental conditions. The N-hydroxylated amines covalently bound to DNA directly or after being acylated with amino acids by amino-acyl-tRNA synthetase. Various species of cytochrome P-450 preparations showed markedly different activity in N-hydroxylation and mutagenic activation of Trp-P-2, Glu-P-1 and IQ. A high spin form of cytochrome P-450, isolated from the liver of PCB-treated rats, showed very high activity in N-hydroxylation of Trp-P-2, Glu-P-1 and 2-aminofluorene, although its activity was very low in benzo(a)pyrene hydroxylation. The present results indicate that different species of cytochrome P-450 are involved in the N-hydroxylation and mutagenic activation of aromatic amines.

Isolation of a high spin form of cytochrome P-450 induced in rat liver by 3-methylcholanthrene

A form of cytochrome P-450 (P-450 MC 1) has been isolated from the livers of 3-methylcholanthrene-treated rats. The molecular weight is 54,500 and the heme iron is in the high spin configuration which clearly differenciates this form from the other major cytochrome induced by 3-methylcholanthrene (P-450 MC 2). Whilst MC 2 actively dealkylated 7-ethoxycoumarin and 7-ethoxyresorufin, MC 1 was only active with 7-ethoxyresorufin. Ouchterlony immunodiffusion analysis and ELISA showed that anti MC 1 and anti MC 2 reacted with both MC 1 and MC 2 but preferentially with the homologous antigen. Both anti MC 1 and MC 2 cross-reacted strongly with microsomes from 3-methylcholanthrene, Aroclor 1254 and isosafrole-treated rats and also, but much weaker, with microsomes from phenobarbital, trans -stilbene oxide and chlofibrate-treated as well as untreated rats. Both MC 1 and MC 2 are induced by the same inducers, 3-methylcholanthrene, Aroclor 1254 and also isosafrole, whilst phenobarbital, trans -stilbene oxide and chlofibrate did not induce either of them, which shows that MC 1 and MC 2 are under similar control by various types of inducers, but MC 1 was present in control microsomes at higher levels than MC 2.

Magnetic and natural circular dichroism of L-tryptophan 2,3-dioxygenases and indoleamine 2,3-dioxygenase. I. Spectra of ferric and ferrous high spin forms.

The ferric form of L-tryptophan 2,3-dioxygenases from both Pseudomonas acidovorans (ATCC 11299b) and rat liver showed magnetic CD spectra ascribable to a high spin protohemoprotein at neutral pH, whereas the ferric indoleamine 2,3-dioxygenase from rabbit intestine exhibited a spectrum due to a mixture of high and low spin states under comparable conditions. Upon addition of L-tryptophan, the spectra of the former enzymes changed to another type of high spin spectra, while the latter showed a marked increase in the low spin component. From these findings and effects of pH on the spectra, it is suggested that the sixth ligand of ferric L-tryptophan 2,3-dioxygenases is water at a neutral pH and that for the ferric indoleamine 2,3-dioxygenase is a strong field ligand such as an imidazole nitrogen. The mixed spin state observed for the latter enzyme was ascribable to a thermal equilibrium between high and low spin states as judged by low temperature spectroscopy. With the ferrous form, the Soret magnetic CD spectra of these enzymes were all similar, giving those of a typical high spin ferrous protohemoprotein, whereas visible spectra were different from one another, suggesting differences in the electronic structure of the heme and its vicinity. The natural CD spectra of both ferric and ferrous forms of each enzyme showed negative Cotton effects in the Soret region. Their intensities were different from one another, presumably due to some differences in the interaction of the heme with nearby aromatic amino acid residue(s).

Proton magnetic resonance spectra of the heme undecapeptide (microperoxidase), its reaction with hydrogen peroxide and its cyanide ligated form

Proton magnetic resonance spectra of the heme undecapeptide (microperoxidase), its reaction with hydrogen peroxide and its cyanide ligated form

Fe(III)·Bleomycin-DNA system. Evidence of Fe(III) to DNA coordination

Bleomycin (BLM) are a family of glycopeptide antibiotics clinically prescribed for the treatment of selected neoplastic diseases [1]. This drug, which both chelates metal ions and binds to DNA, includes a degradation of DNA in a reaction that has been shown to depend, in vitro, on the presence of Fe(II) and molecular oxygen [2]. At the end of the reaction Fe(III)·BLM and degraded DNA are obtained. In this communication we report evidence suggesting that in the BLM·Fe(III)-DNA system, Fe(III) is directly bound to DNA. In Fe(III)·BLM complex four nitrogens, from the secondary amine, the pyrimidine ring, a peptide bond, and the histidine imidazole coordinate to Fe(III) as the basal planar donor; at pH 7 the two axial positions (hereafter labelled A and B) are occupied by the α amino nitrogen and probably an oxygen atom of a glucide, respectively [3]; the complex is then in a low spin form. At pH 4 the α amino nitrogen is no longer bound in A (being presumably superseded by a water molecule) and the metal is therefore in the high spin form. The following experiments suggest that position A can be occupied by different types of ligands and particularly by DNA. 1. at pH 4 by addition of DNA, Fe(III)·BLM is converted into a low spin species which rapidly decays. On the other hand, the addition of ligands (L) such as N − 3, SCN −, S 2O − − 3 to Fe(III)·BLM yields brightly colored complexes in the low spin form. 2. at pH 7 the same colored complexes BLM·Fe(III)·L are obtained by addition of an excess of L. The addition of DNA to these complexes gives rise to the release of L and an BLM·Fe(III)·DNA complex is obtained. These experiments strongly suggest that the various ligands: the α amino group of BLM, N − 3, SCN −, S 2O − − 3 or ADN are competing ligands for the apical position A.

Unusual spin interactions in the 24 heme hydroxylamine oxidoreductase and diheme cytochrome c 554 from nitrosomonas

Nitrosomonas oxidizes NH 3 to HNO 2 with NH 2OH as an intermediate. Oxidation of NH 2OH appears to involve two multiheme cytochromes: hydroxylamine oxidoreductase (HAO) [1] and cytochrome c 554 [2]. Hemes of HAO have midpoint potentials varying from +100 mV to −350 mV [3]. HAO can accept electrons from NH 2OH and pass them to cyt c 554 (midpoint potential −50 mV, 2). HAO, with an α 3β 3 subunit structure, contains 7 c-type hemes and one unique heme P460 per αβ dimer. The CO-binding heme P460 is essential for the NH 2OH dehydrogenase activity and is specifically destroyed by H 2O 2. EPR studies of HAO reveal several classes of low spin (s = 1 2 ) hemes [4]. Two species, accounting for half of the hemes, have been assigned g-values by reductive EPR titration; g = 3.06, 2.14, 1.35 and g = 2.98, 2.24, 1.44 [5]. Only four other EPR signals appear in the oxidized spectrum (g = 3.38, 2.70, 1.85 and 1.66). These resonances titrate coordinately but are not typical of magnetically isolated heme spectra. The apparent g-values of these 4 resonances are frequenCy dependent suggesting that they arise from spin-interactions of the hemes. Frequency dependence of the type observed has not been previously reported. The Mössbauer spectrum of ferric HAO contains a quadrupole doublet at 4.2 K in addition to the expected broad magnetically split spectrum, typical of s = 1 2 hemes. This doublet, which corresponds to at least one and probably two irons per αβ-dimer, has parameters (ΔE Q = 2.1 mm/s and δ Fe = 0.24 mm/s) which are typical of either low spin ferric heme with fast electronic spin relaxation or a pair of spin-coupled hemes [6]. We speculate that this doublet may be associated with the four frequency dependent EPR resonances. Heme P460 is not a component of the latter species since selective destruction of P460 by H 2O 2 fails to alter the EPR spectrum of the oxidized HAO. Thus heme P460 of native HAO is EPR silent. Cytochrome c554 at pH 7 has an unusual 10 K EPR spectrum (g = 4.18, 3.85) similar to intermediate spin (s = 3 2 ) complexes. At pH 4 the EPR spectrum consists of one high spin (g = 6.0, 2.0 and one low spin (g = 2.93, 2.25, 1.52) component. At pH 2 a single high spin component (g = 6.0, 2.0) is present, whereas two low spin forms are observed at pH 10.5. Optical spectra of oxidized cyt c 554 at 20 °C are consistent with high spin heme at pH 4 and low spin heme at pH 10.5. Reduced cyt c 554 reacts with O 2 and binds CO at pH 4: the CO spectrum has two Soret maxima indicating a different interaction with each heme. 1H-NMR spectra at room temperature show contact shifted heme methylene resonances in both the low spin (10–30 ppm) and high spin (60–100 ppm) Fe 3+ spectral regions at all pH values between 4.5 and 9. Contact shifted resonances similar to those reported for s = 3 2 model heme complexes are not observed at this temperature. We conclude that the unusual low temperature EPR spectrum at pH 7 results either from a spin conversion or interaction between high and low spin hemes. EPR, NMR and optical spectra show that this is a different type of heme-heme interaction than observed with diheme cyt c′.