Spectrophotometric investigations with hexa-coordinate ferric lignin peroxidase: does water retention at the active site influence catalysis?

Native lignin peroxidase (LIP) can adopt either a stable penta- or hexa-coordinate state. We have examined catalysis with hexa-coordinate ferric LIP as the starting material, using rapid scanning spectrophotometry. Initial two-electron oxidation of hexa-coordinate native LIP by H(2)O(2) (Compound I formation) was accompanied by a shifting isosbestic point (419-->416 nm), consistent with displacement of a resident water molecule, prior to the reaction of the ferric iron with H(2)O(2). The Compound I species derived from a hexa-coordinate ferric state shows an unusual peak at 520 nm, which may be due to water retention in the vicinity of the heme active site. Compound I reduction by veratryl alcohol showed saturation kinetics, which contrasts with the situation observed when Compound I is derived from a penta-coordinate ferric state. The data inferred that water can interfere with heme access by electron donors, altering the mechanism of Compound I reduction.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

A comparative study of the low-temperature magnetic circular dichroism spectra of horse heart metmyoglobin and bovine liver catalase derivatives

The magnetic circular dichroism (MCD) spectra of three horse heart metmyoglobin compounds, the cyanide, azide and hydroxide forms, have been measured in the visible and near infrared spectral regions at temperatures down to 1.5 K. The three compounds are all virtually completely low-spin at low temperatures with ground g factors of decreasing rhombicity in the order CN- greater than N3- greater than OH-. The MCD magnetization curves have been constructed at selected wavelengths throughout the visible and near infrared regions. The curves are independent of wavelength, showing that all the bands studied are x,y polarized and can, moreover, be satisfactorily fitted to the g factors determined by EPR spectroscopy with theoretical expressions (Thomson, A.J. and Johnson, M.K. (1980) Biochem. J. 191, 411-420). This confirms the assignment and polarizations of the near infrared region low-spin ferric haem charge-transfer bands. The energies of these transitions are markedly dependent upon the added axial ligand, ranging from 1595 to 1295, and 1050 nm for the compounds CN-, N3- and OH-. The MCD spectra of bovine liver catalase and its cyanide adduct have been recorded in the Soret, visible and near infrared regions. Catalase is know to have phenolate anion as the proximal ligand of the haem group. The forms of the spectra make an interesting comparison with those of the analogous metmyoglobin derivatives, in which histidine is the proximal ligand. The MCD spectra of catalase at 4.2 K is an example of a fully high-spin haemoprotein. The cyanide compound is completely low-spin at 4.2 K. The near infrared charge-transfer band is at 1300 nm, showing the effect on the energy of this band of changing from imidazole to phenolate ion as the proximal ligand to haem.     

Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism.

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.     

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.     

Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

Amphitrite ornata dehaloperoxidase (DHP) and Notomastus lobatus chloroperoxidase (NCPO) catalyze the peroxide-dependent dehalogenation of halophenols and halogenation of phenols, respectively. Both enzymes have histidine (His) as their proximal heme iron ligand. Crystallographic examination of DHP revealed that it has a globin fold [M.W. LaCount, E. Zhang, Y.-P. Chen, K. Han, M.M. Whitton, D.E. Lincoln, S.A. Woodin, L. Lebioda, J. Biol. Chem. 275 (2000) 18712-18716] and kinetics studies established that ferric DHP is the active state [R.L. Osborne, L.O. Taylor, K. Han, B. Ely, J.H. Dawson, Biochem. Biophys. Res. Commun. 324 (2004) 1194-1198]. NCPO likely has these same properties. Previous work with His-ligated heme proteins has revealed characteristic spectral distinctions between dioxygen binding globins and peroxide-activating peroxidases. Since DHP, and likely NCPO, is a peroxide-activating globin, we have sought to determine in the present investigation whether the ferric resting states of these two novel heme-containing enzymes are myoglobin-like or peroxidase-like. To do so, we have examined their exogenous ligand-free ferric states as well as their azide, imidazole and NO bound ferric adducts (and ferrous-NO complexes) with UV-Visible absorption and magnetic circular dichroism spectroscopy. We have also compared each derivative to the analogous states of horse heart myoglobin (Mb) and horseradish peroxidase (HRP). The spectra observed for parallel forms of DHP and NCPO are virtually identical to each other as well as to the spectra of the same Mb states, while being less similar to the spectra of corresponding HRP derivatives. From these data, we conclude that exogenous ligand-free ferric DHP and NCPO are six-coordinate with water and neutral His as ligands. This coordination structure is distinctly different from the ferric resting state of His-ligated peroxidases and indicates that DHP and NCPO do not activate bound peroxide through a mechanism dependent on a push effect imparted by a partially ionized proximal His as proposed for typical heme peroxidases.     

Modulation of oxygen binding to insect hemoglobins: the structure of hemoglobin from the botfly Gasterophilus intestinalis

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 A crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O(2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).     

Low-temperature magnetic circular dichroism studies of the photoreaction of horseradish peroxidase compound I

Horseradish peroxidase (HRP) compound I is photolabile at all temperatures between room temperature and 4 K. The photoredox reaction has been studied in frozen glassy solutions by using optical absorption and magnetic circular dichroism spectra following photolysis of HRP compound I with visible-wavelength light at 4.2 and 77 K. The photochemical process is characterized as a concerted two-electron transfer reaction which results in the conversion of the Fe(IV) heme pi-cation radical species of HRP compound I into a low-spin Fe(III) heme species. This reaction occurs even when photolysis is carried out at 4.2 K. Spectra recorded between 4.2 and 80 K for the low-spin ferric hydroxide complex of HRP closely resemble the data measured for the photochemical product. The proposed mechanism for the photoreaction is (formula; see text) No evidence is found for the formation of an Fe(II) heme at these temperatures.     

EPR and ENDOR studies of cryoreduced compounds II of peroxidases and myoglobin. Proton-coupled electron transfer and protonation status of ferryl hemes

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.     

A study of the K+-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side

A series of ferric and ferrous derivatives of wild-type ascorbate peroxidase (APX) and of an engineered K⁺-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4??°C. Wild-type ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five-coordinate high-spin heme ligated on the proximal side by a histidine at pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K⁺-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K⁺-site APX mutant are essentially identical to those of cytochrome b ₅, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K⁺-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K⁺-binding site which is located ～8?Å from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.     

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 of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.     

Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH.

Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70. This behavior is similar to that exhibited by the ferrous form of the enzyme. However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP. Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70. In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min). F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction. Horseradish peroxidase, however, does not. The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket. Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme. We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

Substitution of strictly conserved Y111 in catalase-peroxidases: Impact of remote interdomain contacts on active site structure and catalytic performance

Catalase-peroxidase function is strictly dependent on a gene-duplicated C-terminal domain. This domain no longer has a functioning active site, but from 25 to 30 Å away it is essential for preventing the coordination of an active site base (His106) to the heme. The mechanisms by which this distant structure supports active site function have not yet been elucidated. Tyr111 is a strictly conserved member of an interdomain H-bonding network that supports the loop connecting the N-terminal B (bearing His106) and C helices. Spectroscopic evaluation of the Tyr111Ala variant of KatG showed a substantial increase in hexa-coordinate low-spin heme, giving it the appearance of a transition between the wild type (primarily high-spin) and the N-terminal domain alone (pure low-spin). Concomitant with the spectral changes was decreased activity compared to the wild type enzyme, suggesting that Tyr111 does have a role in preventing His106 coordination. Substitution of Tyr111 diminishes catalase activity more substantially than peroxidase activity. Such an effect cannot be explained by His106 coordination alone, suggesting that these interdomain interactions may help tune the catalase-peroxidase active site for bifunctionality.     

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.     

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.     

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.     

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.