Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.     

Heme reactivity of hemoglobins. Azide and fluoride binding equilibria of free and mercuriated ferri-gamma chains.

The free gamma chains, isolated from human foetal hemoglobin, are stable when oxidized and thus suitable for ligand binding and subunit equilibrium studies. The metaquo-ferri chains, with cysteine-F9 in the free state II ag gamma SH) possess several properties which are different from those of their p-mercuribenzoate derivative (III aq gammaSHgR); these are: stronger binding of a high-field ligand (N3- minus), altered spin equilibrium and an altered subunit equilibrium. A quantitative assessment of the free energy changes associated with all individual steps involved in changing the metaquo chains to their azide derivatives has been made. The results show that the higher apparent reactivity of III ag gammaSH (compared to IIIaq gammaSHgR) for the azide ion is not solely due to compensatory effects arising from differences of subunit dissociation or of spin equilibrium: other process(es) occurring in the ligand binding site have to be considered.     

Azide binding to the cytochrome c ferric heme octapeptide. A model for anion binding to the active site of high spin ferric heme proteins.

Equilibrium constants for the binding of azide to the ferric heme c octapeptide in 50% ethylene glycol 50% buffer were measured spectrophotometrically. The equilibrium constant for azide binding at 20 degrees C and pH* 7.4 is 29.2, which is approximately 3 to 4 orders of magnitude lower than that observed for azide binding to various ferric hemeproteins. The equilibrium constant was indepent of pH* in the range from 7 to 8. Equilibrium constants at several temperatures exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta H0 = -26,100 J/mol (-6240 cal/mol) and delta S0 = -61.5 J/0K mol (-14.7 e.u.). Comparison of these values to the values for the heme proteins enables one to explain the differences in equiliberium constants in terms of differences in the polarity of the heme environments. The results are consistent with the concept that the oxygen affinity of heme complexes increases with the polarity of the heme environment. The data also suggest that an increase in the polarity of the heme environment should result in a corresponding increase in the susceptibility of ferrous heme dioxygen complexes toward oxidation by the dioxygen.     

Azide binding to the trinuclear copper center in laccase and ascorbate oxidase.

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1:1 to 1:7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximately 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximately 1.97 signal, with similar saturation characteristics was found in the AO : azide (1:7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti-ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1:7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g parallel region that are attributed to a perturbed T2 in protein molecules where the anti-ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6-90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.     

Influence of quaternary structure of the globin on thermal spin equilibria in different methemoglobin derivatives.

We have measured the paramagnetic susceptibilities of sperm whale azide metmyoglobin and of carp azide, thiocyanate, and nitrite methemoglobin in the quaternary oxy (R) and deoxy (T) structures between about 300 and 90 K, using a new sensitive superconducting magnetometer. We have also measured the pressure dependence of the high- and low-spin optical absorption bands of azide metmyoglobin and of carp azide methemoglobin in the R and T structures between 1 and 2000-4000 atmospheres. At low temperatures all the derivatives show normal Curie behavior, but above 200-250 K this is reversed, so that a thermal spin equilibrium is set up and the paramagnetic susceptibilities rise steeply with rising temperature. At all temperatures the effective magnetic moments in the T structure are higher than in the R structure. The magnetic data for azide methemoglobin have been subjected to detailed analysis. Below 250 K the magnetic moment in the R structure is 1.98 microB, characteristic of pure low spin, but that in the T structure is 2.80 microB, suggestive of a random mixture of high- and low-spin centers which have become frozen in by the immobility of the surrounding protein. Comparison of the thermal spin equilibria above 250 K shows that in the T structure the equilibrium is biased toward higher spin by the equivalent of about 1 kcal/mol relative to the R structure. Hydrostatic pressure reduces the optical density of the high-spin band at 630 nm and increases that of the low-spin bands at 541 and 573 nm. We have calibrated the optical density of the band at 630 nm against the measured paramagnetic susceptibilities of sperm whale azide metmyoglobin and carp azide methemoglobin in the R and T structures and have used this calibration to determine the dependence of the spin equilibria on hydrostatic pressure; this has allowed us to calculate the volume contraction associated with the transition from the fully high to the fully low-spin state. This amounts to -6.7 and -13.3 mL/mol heme for carp azide methemoglobins in the R and T structures, respectively, and to -12.5 mL/mol heme for azide metmyoglobin. These volume contractions are larger than those of about -4 mL/mol Fe found in synthetic iron chelates. Apparently stereochemical changes of the globin surrounding the heme also contribute to the volume changes; these must be larger in the T than in the R structure. The significance of these observations for the mechanism of heme-heme interaction is discussed.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition.

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represent a3+(3) (Beinert, H. and Shaw, R.W.(1977) Biochim. Biophys. Acta 462, 12u--130), and can be detected and quantitatively determined by EPR. We have now used out ability to generate this species to study reactions of a3+(3) with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O2 and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a3+(3) reacts within less than 10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a3+(3) species responds to O2 and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a3+(3) registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a3+(3) in either case. In all these experiments, in which EPR detectable a3+(3) was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a3+(3) to other 'activated' species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.     

Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin.

Vanadyl ion, VO(IV), has been used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of human serum transferrin in the presence of bicarbonate. Iron-saturated transferrin does not bind the vanadyl ion. Room temperature titrations of apotransferrin with VO(IV) as monitored by EPR indicate the extent of binding to be pH dependent, with a full 0.2 VO(IV) ions per transferrin molecule bound at pH 7.5 and 9, but only about 1.2 VO(IV) ions bound at pH 6. The EPR spectra of frozen solutions with or without 0.1 M NaCUO4 at 77 K show that there are two spectroscopically nonequivalent binding sites (A and B) with a slight difference in binding constants. One site (A site) exhibits essentially constant binding capacity in the pH range 6-9, but the other (B site) becomes less avialable as the pH is reduced below 7. Results with mixed Fe(III)-VO(IV) transferrin complexes suggest that iron shows a slight tendency to bind at the B site over the A site pH 7.5 and 9.0. Only the B site in both vanadyl and iron transferrins is perturbed by the presence of perchlorate.     

Calcium-induced cooperativity of manganese binding to concanavalin A.

Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.     

Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules

Conformational changes induced by ligands and pH in lupine ferrileghemoglobin selectively modified at Tyr-E16 by the imidazolide spin label has been studied by the method of electron spin resonance in the pH range 6-13. It is shown that in the alkaline pH region the bound spin label registers a local conformational transition which precedes the alkaline denaturation of the protein. In aquamet, cyanide and nicotinate complexes of ferrileghemoglobin this transition occurs with pK 10.5, in acetate and azide complexes with pK 11. In all these ligand derivatives the transition is induced by alteration in the ionization state of one group (delta nH+ approximately equal to 1), most probably, the epsilon-amino group of Lys-GH3. The latter is linked with the Glu-A14 residue and this bond is essential for maintaining the native conformation of leghemoglobin. The ligand-induced conformational changes in the vicinity of the label are small and consist, most likely, in some alteration of the mutual arrangement of the AE and GH helical complexes. No correlation has been revealed between the spin state of the heme iron and the conformation of leghemoglobin in the region under study.     

The binding of azide to human methemoglobin A0. Error analysis for the interpolative and noninterpolative methods.

The binding azide to human methemoglobin A0 has been studied at 6 degrees, pH 7, and I = 0.2 by three spectroscopic methods: (1) the conventional interpolative method, (2) an interpolative dialysis technique, and (3) a noninterpolative method. The interpolative methods assume that the fractional spectral change equals the fraction of heme sites bound by ligand, while the noninterpolative method measures the extent of binding directly, i.e., without the interpolative assumption. Both experiment and error analysis show that method 1 has low precision, and consequently, gives an inherently unreliable binding isotherm. Method 2 achieves high experimental and intrinsic precision. However, method 3, which also has high precision, clearly proves that the interpolative assumption of method 2 is incorrect. That is, the true fractional extent of binding becomes equal to the fractional spectral change only after about 97% of heme sites have been bound with ligands.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase

The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR). Intensities of the characteristic bands for the high spin heme b, that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (-) in the MCD spectrum decreased considerably. Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g approximately 6, of which less than 5% had been EPR detectable at pH 7. Cyanide (x40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (x1000) was necessary to bind to the reduced NOR at an acidic pH, ca. 5. Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.     

Evidence that azide occupies the chloride binding site near the manganese cluster in photosystem II

The effect of adding azide to photosystem II (PS II) membrane samples (BBY preparation), with or without chloride, has been investigated using continuous wave (CW) and pulsed EPR spectroscopy. In the BBY samples with 25 mM chloride, we observed that the inhibition induced by azide is partly recovered by the addition of bicarbonate. Electron spin-echo envelope modulation (ESEEM) was used to search for spin transitions of 15N nuclei magnetically coupled to the S2 state Mn cluster (multiline EPR signal form) in 15N (single terminal label) azide-treated samples with negative results. However, an 15N ESEEM peak was observed in parallel chloride-depleted PS II samples when the 15N-labeled azide is added. However, this peak is absent in chloride-depleted samples incubated in buffer containing both chloride and [15N]azide. Thus these results demonstrate an azide binding site in the immediate vicinity of the Mn cluster, and since this site appears to be competitive with chloride, these results provide further evidence that chloride is bound proximal to the Mn cluster as well. Discussion on the possible interplay between azide, chloride, and bicarbonate is provided.     

Quaternary structure of methemoglobin. Pulse radiolysis study of the binding of oxygen to the valence hybrid.

The pulse radiolysis of solutions of adult human methemogolbin was used in order to reduce a single heme iron within the protein tetramers. The valence hybrids thus formed were reacted with oxygen. Kinetics of the reactions were studied. The effects of pH and inositol hexaphosphate were examined. The kinetics of the ligation of oxygen to stripped valence hybrids showed a single phase behavior at the pH range 6.5 to 9. As the pH was lowered below 6.5, a second, slower phase became apparent. In the presence of inositol hexaphosphate, above pH 8, the kinetics of oxygen binding was of a single phase. As the pH was lowered, a transition to a second, slower phase was noticed. Below pH 7, the slower phase was the only detectable one. The analysis of the relative contribution of the faster phase to the total reaction as a function of the pH showed a typical transition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9. On this basis, it is concluded that human adult stripped methemoglobin resides in an R quarternary structure, while the presence of IHP stabilizes the T structure at pH below 7.5. This transition between the quaternary structures of methemoglobin cannot be accounted for by the switch between the high spin and the low spin states of the ferric iron. This switch of spin state takes place at pH greater than 8.2.     

Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus

The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.     

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.     

Temperature dependence in the absorption spectra of beef liver catalase

The spin characteristics of the ferric heme groups in native beef liver catalase. and in the complexes formed by reaction with fluoride, cyanide, azide, thiocyanate, and cyanate ions have been studied using absorption spectroscopy over the temperature range of 4–285 K. The azide. isothiocyanate, and isocyanate complexes of catalase are considered to be high-spin ferric heme complexes at room temperature, but undergo a thermal spin change below 300 K. The temperature dependence of these absorption spectra, however, cannot be analyzed in terms of simple Boltzmann distributions between two S = $\text{1}\text{2}$ and S = $\text{5}\text{2}$ spin stales. The data show that these spin changes occur over a very narrow temperature range, but do not result in the formation of completely, low-spin complexes. The data also suggest that the thermal spin changes that occur below the glassing temperature of the solvent are dependent upon the conformational changes which take place within the protein itself with a change in temperature, and which directly affect the environment of the heme group.