NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.     

1H NMR spectroscopy of Paracoccus denitrificans cytochrome c-550

The 1H NMR spectra of ferri- and ferro-cytochrome c-550 from Paracoccus denitrificans (ATCC 13543) have been investigated at 300 MHz. The ferri-cytochrome c-550 shows hyperfine-shifted heme methyl resonances at 29.90, 29.10, 16.70, and 12.95 ppm and a ligand methionyl methyl resonance at -15.80 ppm (pH 8 and 23 degrees C). Four pH-linked structural transitions were detected in spectra taken as a function of pH. The transitions have been interpreted as loss of the histidine heme ligand (pK less than or equal to 3), ionization of a buried heme propionate (pK = 6.3 +/- 0.2), displacement of the methionine heme ligand by a lysyl amino group (pK congruent to 10.5), and loss of the lysyl ligand (pK greater than or equal to 11.3). The temperature behavior of hyperfine-shifted resonances was determined. Two heme methyl resonances (at 16.70 and 12.95 ppm) showed downfield hyperfine shifts with increasing temperature. The cyanoferricytochrome had methyl resonances at 23.3, 20.1, and 19.4 ppm. NMR spectroscopy did not detect the formation of a complex with azide. The second-order rate constant for electron transfer between ferric and ferrous forms was determined to be 1.6 X 10(4) M-1 s-1. Heme proton resonances were assigned in both oxidation states by cross-saturation and nuclear Overhauser enhancement experiments. Spin-coupling patterns in the aromatic region of the ferro-cytochrome spectrum were investigated.     

Proton NMR spectroscopy of cytochrome c-554 from Alcaligenes faecalis

Cytochrome c-554 from the bacterium Alcaligenes faecalis (ATCC 8750) is a respiratory electron-transport protein homologous to other members of the cytochrome c family. Its structure has been studied by 1H NMR spectroscopy in both the ferric and ferrous states. The ferric spectrum is characterized by downfield hyperfine-shifted heme methyl resonances at 46.25, 43.60, 38.40, and 36.73 ppm (25 degrees C, pH 7.1). Chemical shifts of these resonances change with temperature opposite to expectations derived from Curie's law. The pH behavior of the hyperfine-shifted resonances titrates with a pK of 6.3 that has been interpreted as due to ionization of a heme propionate. In the ferrous state, heme methyl, meso, and thioether bridge resonances have been observed and assigned. All aromatic proteins have been assigned according to the side chain of origin, and the structural environment about the sole tryptophan residue has been examined. The electron-transfer rate between ferric and ferrous forms has been estimated to be on the order of 3 X 10(8) M-1 s-1, which is the largest such self-exchange rate yet observed for a cytochrome.     

Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5 x 10(5) M-1) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5-10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x 10(6) M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.     

Proton magnetic resonance spectra of adrenodoxin: features of the aromatic region

This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.     

Proton NMR spectroscopy of Alcaligenes cytochrome c556

A high molecular-weight c-type cytochrome was purified from Alcaligenes faecalis ATCC 8750. Its weight was 40,000 daltons by sodium dodecyl sulfate-gel electrophoresis. Heme content was determined to be one heme per 40,000 daltons. Proton nuclear magnetic resonance-NMR-spectroscopy determined that the ferrous form is low spin. The detection of a methyl resonance at -3 ppm in the ferrous form indicated that methionine is a heme ligand in this state. The NMR spectrum of the ferric form at pH 7.2 revealed hyperfine shifted methyl resonances at 67.79, 63.17, 57.71, and 50.46 ppm. The large downfield shifts observed are indicative of high spin character. The ferric spectrum was pH-sensitive, indicating two pH-linked structural transitions with estimated pKs at 6.0 and 10.5. The first is interpreted as due to the ionization of a heme propionate. The second is interpreted as the acquisition of a strong field ligand and the subsequent conversion to a low spin ferric form. The ferricytochrome did not form complexes with cyanide, azide, or fluoride at pH 5.2 or 7.9.     

Preliminary 1H-NMR studies of the interaction between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway

The complex formation of two electron transfer proteins, cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway, has been shown by 1H-NMR spectroscopy. Presence of ferredoxin I produces ferricytochrome c3 1H-NMR spectrum modifications. The chemical shift of perturbated heme methyl resonances has been used to determine the stoichiometry of the complex. At pH 7.6 and 20 degrees C, the two proteins were found to form a complex 1:1 with an association constant, KA, of 10(4) M-1. Two of the four hemes are affected by presence of ferredoxin I and may be involved in the electron transfer sites. The heme methyl resonances are average resonances of free and bound cytochrome c3 resonances, indicating a fast exchange process on the NMR time scale.     

1H-NMR investigation of yeast cytochrome c. Interaction with the corresponding specific reductase (L-lactate cytochrome)

1H-NMR spectroscopy has been used to study the modifications of certain characteristic resonances of the Hansenula anomala yeast cytochrome c on binding to its specific reductase (flavocytochrome b2) or to the isolated cytochrome domain obtained from the entire molecule. Normal titration curves are observed for the resonances at 37.8 ppm assigned to heme c methyl 8 and at 19.4 ppm, line of cytochrome b2 spectrum. In contrast, the shifts near 3.2 and 3.4 ppm for trimethyl-lysine resonances of this cytochrome c present abnormal titration curves, saturation being apparently reached at low molar (cytochrome b2)/(cytochrome c) ratio. An interpretation is proposed in terms of shifts due to local conformational transitions induced by reductase binding but not rapidly reversible upon dissociation.     

Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR

The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand tau nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine phi nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine tau NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the omega loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center.(ABSTRACT TRUNCATED AT 250 WORDS)     

A change in the heme stereochemistry of cytochrome c upon addition of sodium dodecyl sulfate: electron paramagnetic resonance and electronic absorption spectral study

Electron paramagnetic resonance and electronic absorption spectral changes upon addition of sodium dodecyl sulfate (SDS) to ferric and ferrous cytochrome c have been measured at 77 degrees K and at room temperature. The spectral changes upon addition of SDS to ferric cytochrome c were performed, in two steps, from native low-spin to another low-spin spectrum and subsequently to high-spin-like spectrum. On the other hand, the spectral changes upon addition of SDS to ferrous cytochrome c proceeded, in one step, from native low-spin to high-spin spectrum. The high-spin-like spectrum of ferric cytochrome c and the high-spin spectrum of ferrous cytochrome c in the presence of high concentrations of SDS are, respectively, apparently similar to those of ferric and ferrous cytochrome c' at physiological pH in spectral features. These spectral similarities suggest the similarities in the heme stereochemistry and the ground state of heme iron. Further, the spectra of cytochrome c in the presence of SDS varied with the change of pH values. The ferric high-spin-like and ferrous high-spin spectra were stable at neutral pH and below it. Conformational changes of cytochrome c upon addition of SDS are also discussed.     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.     

Proton NMR spectroscopy of sulfmyoglobin

The 1H NMR spectra of ferrous sulfmyoglobin, metsulfmyoglobin, and ferric cyanosulfmyoglobin were obtained at 300 MHz. Hyperfine-shifted resonances are observed in the case of metsulfmyoglobin and ferric cyanosulfmyoglobin that have line widths and cover a chemical shift range that are comparable to the corresponding forms of normal myoglobin. Two methyl resonances are observed in the spectrum of ferric cyanosulfmyoglobin at 44.19 and 25.48 ppm (25 degrees C, pH 8.3) that have been assigned to heme methyls at the 8- and 5-positions on the basis of pH titration effects homologous to the corresponding methyl resonances in ferric cyanomyoglobin. Examination of aromatic region resonances and the pH titration profiles of histidine resonances lead to the conclusion that the overall conformation of sulfmyoglobin was highly homologous to that of normal myoglobin and afforded assignments of histidine residues of the former. The most likely position for the addition of a sulfur atom to the heme of sulfmyoglobin is pyrrole ring A, with ring B a possible, but less likely, alternative.     

Resonance Raman characterization of Chromatium vinosum cytochrome c'. Effect of pH and comparison of equilibrium and photolyzed carbon monoxide species

Resonance Raman spectra of Chromatium vinosum cytochrome c' have been obtained for the five pH-dependent states of the protein [i.e., types I (pH 7), II (pH 10), and III (pH 12) of the ferric protein and type a (pH 7) and type n (pH 12) of the ferrous protein]. The raman spectra of type II and type a are consistent with those of high-spin, 5-coordinate heme proteins, such as deoxyhemoglobin, while spectra of type III and type n correspond more closely to those of low-spin, ferric and ferrous cytochrome c, respectively. Spectra of the CO-bound equilibrium species qualitatively resemble those of carbon monoxy human HbA. However, both the Fe-C and C = O stretching modes of the ligated species exhibit pH-dependent frequency shifts. Our data also indicate that CO photolysis is much more efficient at pH 7 than at pH 12. Moreover, the spectra of the photolytic transients suggest that unique, high-spin species are formed subsequent to CO photolysis from both type a and type n species.     

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.     

A nuclear magnetic resonance study of axial ligation for the reduced states of chloroperoxidase,cytochrome P-450cam,and porphinatoiron(II) thiolate complexes

The reduced forms of cytochrome P-450_cam and chloroperoxidase were examined by proton NMR spectroscopy. The pH and temperature dependences of the proton NMR spectra of both ferrous enzymes are reported. A series of alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins was also examined. The proton NMR spectra of these complexes facilitated the assignment of resonances due to the axial ligand in the model compounds on the basis of their isotropic shifts and multiplicities. Comparison of model compound data with that for the reduced enzymes supports assignment of the methylene protons for the axial cysteinate of ferrous cytochrome P-450_cam and ferrous chloroperoxidase to proton NMR resonances at 279 and 200 ppm (pH 7.0, 298K), respectively. Differences in the active site structure of the two enzymes are further demonstrated by ¹⁵N-NMR spectroscopy of the cyanide complexes of the ferric forms.     

13C-NMR spectroscopy of acetyltyrosyl-guanidinated horse heart cytochrome c

The tyrosine residues of guanidinated horse heart cytochrome c have been specifically acetylated by reaction with N-[1-13C]acetylimidazole (90 atom%). Acetylation was monitored by 13C-NMR spectroscopy. The tyrosine residues were found to show widely varying reactivities ranging from one that is completely and exclusively acetylated at low reagent concentration (residue 67) to one that is acetylated only when the protein is unfolded (residue 97). Homogeneous derivatives were prepared containing one (either residue 67 or 97), three 48, 67 and 74), or four (residues 48, 67, 74 and 97) O-[1-13C]acetyl groups. 13C-NMR spectra of selected derivatives were obtained at pH 5.8, in the presence of cyanide ion, in the ferrous and ferric oxidation states, and after denaturation with 6M guanidine hydrochloride. The O-[1-13C]acetyltyrosyl resonances gave chemical shift values ranging from 171.8 to 176.0 ppm. These resonances were assigned to specific groups based on the known order of reactivity of the tyrosyl side chains toward N-acetylimidazole. The chemical shift of O-[1-13C]acetyltyrosyl 67 was found to be particularly sensitive to changes in protein structure. The proximity of this group to the heme makes it subject to distance-dependent paramagnetic and ring current effects. Acetylation of tyrosyl 74 gives rise to a pH-dependent equilibrium between conformers in the ferric state and a conformation change in the ferrous state. Acetylation of this residue also leads to an absorbance decrease at 695 nm that can be related to the 13C-NMR-detected conformational equilibrium. Addition of cyanide ion abolished this equilibrium.     

1H NMR structural characterization of the cytochrome c modifications in a micellar environment

The interaction of cytochrome c with micelles of sodium dodecyl sulfate was studied by proton NMR spectroscopy. The protein/micelles ratio was found to be crucial in controlling the extent of the conformational changes in the heme crevice. Over a range of ratios between 1:30 and 1:60, the NMR spectra of the ferric form display no paramagnetic signals due to a moderately fast exchange between intermediate species on the NMR time scale. This is consistent with an interconversion of bis-histidine derivatives (His18-Fe-His26 and His18-Fe-His33). Further addition of micelles induces a high-spin species that is proposed to involve pentacoordinated iron. The resulting free binding site, also encountered in the ferrous form, is used to complex exogenous ligands such as cyanide or carbon monoxide. Attribution of the heme methyls was performed by means of exchange spectroscopy through ligand exchange or electron transfer. The heme methyl shift pattern of the micellar cyanocytochrome in the ferric low spin form is different from the pattern of both the native and the cyanide cytochrome c adduct, in the absence of micelles, reflecting a complete change of the heme electronic structure. Analysis of the electron self-exchange reaction between the two redox states of the micellar cyanocytochrome c yields a rate constant of 2.4 x 10(4) M(-1) s(-1) at 298 K, which is surprisingly close to the value observed in the native protein.     

Magnetic susceptibility measurements on Pseudomonas cytochrome cd1

The magnetic susceptibilities of cytochrome cd1 from Pseudomonas aeruginosa (American Type Culture Collection 19429) have been measured by a nuclear magnetic resonance technique. In the oxidized form both heme c and heme d1 are in the low-spin state with an average magnetic moment of 2.6 Bohr magnetons. At 25 degrees C and pH 8.0, the ascorbate-reduced cytochrome contains one low-spin and one high-spin heme per subunit. Based on previous reports in the literature, the high-spin ferrous heme has been assigned to the heme d1 group. At pH 8.0 the ascorbate-reduced heme d1 has a magnetic moment of 5.3 Bohr magnetons. This value decreases to 4.9 at pH 5.5, but is still indicative of a high-spin ferrous system. The paramagnetic susceptibility of the ferricytochrome demonstrated a temperature dependence consistent with Curie's law, but the ferrocytochrome showed an increase in paramagnetic susceptibility with increasing temperature.