Myeloperoxidase of the leukocyte of normal blood. Nature of the prosthetic group of myeloperoxidase

The absorption spectra of alkaline pyridine hemochrome of myeloperoxidase in its native, acid, and modified forms were similar to those of heme a, and the molar extinction coefficient of myeloperoxidase heme was very similar to that of heme a, assuming that myeloperoxidase contains only one heme. The anaerobic titration of myeloperoxidase with dithionite showed that one electron was consumed per molecule of the enzyme for its conversion to its reduced form. The EPR spectrum of myeloperoxidase indicated that the enzyme contains both high-spin heme and non-heme iron. Carbonyl reagents, such as borohydride, hydrazine, and benzhydrazide, reacted with myeloperoxidase, causing blue shifts in its absorption spectrum. The heme was labeled with a tritium of boro[3H]hydride, suggesting that the reagents reacted with a formyl group on the porphyrin ring of the myeloperoxidase heme. When hydrazine was added to cyanide complex I of myeloperoxidase the complex was converted to the hydrazine-enzyme compound. Myeloperoxidase reacted with bisulfite to form a compound with an absorption spectrum similar to that of cyanide complex I. Borohydride-treated myeloperoxidase formed only one cyanide complex, while the native enzyme formed two different cyanide complexes, I (Kd = 0.3 muM) and II (approximate Kd = 0.1 mM). The EPR spectrum indicated that cyanide complex I of myeloperoxidase still contained high-spin heme. The results suggested that cyanide complex I and the bisulfite compound of myeloperoxidase were adducts between the nucleophilic reagents and the formyl group of myeloperoxidase heme. Based on these results, we concluded that one of the two iron atoms in a myeloperoxidase molecule exists in a formyl-heme moiety similar to heme a and the other exists as a non-heme iron.     

Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.     

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.     

Purification to homogeneity and initial physical characterization of secondary amine monooxygenase

Secondary amine monooxygenase from Pseudomonas aminovorans grown on trimethylamine has been purified 265-fold to apparent homogeneity. The purified enzyme exhibits a specific activity of 14.7 mumol of NADPH oxidized per min per mg of protein, a native molecular weight of 210,000, and nondisulfide-linked subunits of molecular weight 42,000, 36,000, and 24,000, each of which is required for activity. The enzyme is extremely labile during purification; rapid handling and the presence of 5% ethanol are essential to enzyme stability. Storage at 77 K in the presence of NADH (1 mM) also confers considerable stability to the purified enzyme. The heme prosthetic group in the enzyme has been identified as protoporphyrin IX. The quantification of prosthetic group components reveals the presence of 1.6 mol of flavin as FMN, 2.0 mol of heme iron, 4.0 mol of acid-soluble (nonheme) iron, and 3.6 mol of free sulfide/210,000 molecular weight enzyme. Ferric and ferrous-CO secondary amine monooxygenase exhibit electronic absorption spectra that are very similar to those of analogous myoglobin derivatives and, therefore, quite distinct from parallel forms of cytochrome P-450, the most extensively studied heme iron-containing monooxygenase. Like myoglobin and, again, in contrast to P-450, this enzyme forms a very stable dioxygen complex. In fact, it is this oxygen-bound form of the enzyme that is obtained from the purification procedure. Once again, the absorption spectrum of oxygenated secondary amine monooxygenase is nearly identical to that of oxymyoglobin. The spectroscopic similarities between secondary amine monooxygenase and myoglobin suggest the presence of an endogenous histidine fifth ligand to the heme iron of the enzymes.     

Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

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.     

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.     

Photochemically modified myeloperoxidase, with optical spectral properties analogous to those of lactoperoxidase, retains its original catalytic activity

During the course of a reducing reaction using ketyl radicals generated from ketone photoreduction with ultraviolet light, a photoinduced chemical modification of the chromophore group in myeloperoxidase has been found. Light absorption and resonance Raman spectra for this modified enzyme indicated an iron porphyrin chromophore group. The alkaline pyridine hemochrome of the modified enzyme exhibited an optical spectrum closely related to that of iron protoporphyrin IX. The chromophore group of the modified myeloperoxidase was cleaved from the protein by methoxide. Proton magnetic resonance of the diamagnetic bis(cyanide) compound of the extracted heme group showed the presence of two vinyl and three methyl side chains associated with a porphyrin macrocycle. These data provide further insight into the structure of the active site in myeloperoxidase. The EPR spectral properties and enzymatic activities of the native myeloperoxidase are essentially conserved in the modified enzyme. Our present results indicate that the heme peripheral substituent is modified while the stereochemical structure surrounding the chromophore group is not altered by the photochemical modification.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

Resonance Raman studies of lactoperoxidase

Resonance Raman (RR) spectra obtained at three excitation wavelengths are reported for various ferric, ferrous, and ferryl derivatives of bovine lactoperoxidase. The RR spectra of the ferric derivatives show the full complement of the vinyl stretching and scissor modes indicating that the two vinyls in the protoporphyrin IX prosthetic group are present in unmodified forms. The cysteine thiol complex exhibits a RR spectrum identical to that of the native enzyme, an observation which strongly suggests a nonheme binding site for the thiol substrates. The different ferrous complexes of lactoperoxidase which result from heme reduction at slightly alkaline and acidic pH gave identical low-frequency RR spectra. Differences are observed, however, in the high-frequency region. Reduction in the presence of cyanide, however, yields two time-resolved complexes. Changes in the ligand field during the conversion to the final form of the cyanoferrous complex are proposed based on the changes observed in the low-frequency vibrational spectrum. Comparisons are made between the low-frequency RR spectra of the limiting form of the cyanoferrous and the nitric oxide lactoperoxidase complexes. The similarity between the RR spectra of these two complexes in the 150-500 cm-1 region supports the assignment of structures for these complexes where the six-coordinate heme iron is displaced from the heme plane and away from the proximal histidine ligand.     

Presence of endogenous calcium ion and its functional and structural regulation in horseradish peroxidase

The endogenous calcium ion (Ca2+) in horseradish peroxidase (HRP) was removed to cause substantial changes in the proton NMR spectra of the enzyme in various oxidation/spin states. The spectral changes were interpreted as arising from the substantial alterations in the heme environments, most likely the heme proximal and distal sides. The comparative kinetic and redox studies revealed that these conformational changes affect the reduction process of compound II, resulting in the decrease of the enzymatic activity of HRP. It is also revealed from the ESR spectrum and the temperature dependences of the NMR and optical absorption spectra of the Ca2+-free enzyme that the heme iron atom of the Ca2+-free enzyme is in a thermal spin mixing between ferric high and low spin states, in contrast to that of the native enzyme. These results show that Ca2+ functions in maintaining the protein structure in the heme environments as well as the spin state of the heme iron, in favor of the enzymatic activity of HRP.     

Heme maintains catalytically active structure of cytochrome P-450

Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apoand reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.     

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Magnetic circular dichroism studies of bovine liver catalase.

Absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of beef liver catalase at pH 5.0 and 6.9, and its complexes with NaF, KCNO, NaCNS, NaN3 and NaCN, have been measured between 250 nm and 700 nm at room temperature. The pH 6.9 native catalase MCD shows the presence of several additional transitions not resolved in the absorption spectrum. While these bands can be seen in the spectra of all the derivatives, with the exception of the cyanide, their relative intensities changes considerably between complexes. Of special interest in the MCD of ferric hemes is the signal intensity at about 400 nm and 620 nm. The data indicate that the MCD intensity at 620 nm increases as the high spin iron porphyrin fraction increases, reaching a maximum with the fluoride complex. The 430 nm band intensity increases as the proportion of low spin iron increases, reaching a maximum with the cyanide complex. The MCD spectra also indicate clearly the existence of spin mixtures in the complexes with CNO-, CNS-, and N3-, where both the 430 nm and 620 nm bands have appreciable intensity. It is significant that despite almost identical absorption spectra the CNS- complex has higher fraction of low spin iron than either the CNO- or the N3- species. The differences between the pH 5 and 6.9 MCD spectra of the native catalase suggest that the environment of the heme centre is sensitive to protonation.     

Conformational analysis of hemopexin by fourier-transform infrared and circular dichroism spectroscopy

Hemopexin is a serum glyco-protein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β-structure, 15% α-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme–hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis. © 1994 Wiley-Liss, Inc.     

Circular dichroism studies of low-spin ferric cytochrome P-450CAM ligand complexes

Circular dichroism (CD) spectroscopy has been used to probe the active site of bacterial ferric cytochrome P-450CAM. The endogenous sixth ligand to the heme iron has been displaced by an extensive series of exogenous oxygen, nitrogen, sulfur and other neutral and anionic donor ligands in an attempt to examine systematically the steric and electronic factors that influence the coupling of the heme chromophore to its protein environment. General trends for each ligand class are reported and discussed. Both the wavelengths and the intensities of the CD bands vary with ligand type and structure. All but one of the complexes exhibit negative CD maxima in their delta and Soret bands. Comparison to ferric myoglobin-thiolate complexes indicates that the negative sign observed for the cytochrome P-450 spectra is not a property of the thiolate fifth ligand, but rather arises from a different interaction of the cytochrome P-450 heme with its protein environment. Complexes with neutral oxygen donors display CD spectra that most closely resemble the spectrum of the native low-spin enzyme. Hyperporphyrin (split Soret) cytochrome P-450 complexes with thiolates, phosphines and cyanide trans to cysteinate have complex CD spectra, reflecting the intrinsic non-degeneracy of the Soret pi pi transitions. The extensive work presented herein provides an empirical foundation for use in analyzing the interaction of heme chromophores with their protein surroundings, not only for the cytochrome P-450 monooxygenases but also for heme proteins in general.