Soret Magnetic Circular Dichroism Research Articles

Determination of zero-field splitting and evidence for the presence of charge-transfer transitions in the Soret region of high-spin ferric hemoproteins obtained from an analysis of low-temperature magnetic circular dichroism

Theoretical expressions for magnetic circular dichroism (MCD) of the porphyrin π→π*, spin-allowed charge transfer (CT) and spin-forbidden d- d or CT transitions in high-spin ferric heme are derived. The transitions can be discriminated by their MCD to absorption ratio and/or temperature dependence of MCD intensity. An analysis of the Soret MCD of fluoride complexes of myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), recorded at temperatures from 290 down to 2 K, is given. It is shown that the Soret MCD of HRPF can be well described by overlapping of the π→π* transition with one spin-forbidden CT transition of an 6 A 1→ 4 E type. In the case of MbF and HbF it is necessary to assume the presence in the Soret region of the second spin-forbidden CT transition, most probably of an 6 A 1→ 4 A 1 type. The parameters of transitions have been extracted from a non-linear least-squares fitting procedure. The best fit values of parameter D of the zero-field splitting of the ground manifold for HbF (6.1 cm −1) and MbF (6.4 cm −1) agree well with those obtained by other methods. The D value for HRPF (8.3 cm −1) is obtained for the first time.

Insight into externally bound 5,10,15,20-tetrakis(2-N-methylpyridyl)porphyrinatopalladium(II), PdP(2), with B-form DNA duplexes poly(G-C)2, poly(A-T)2, and CT DNA by using combined MCD, CD, and optical data

The Soret (B0) region of free and externally DNA-bound 5,10,15,20-tetrakis(2-N-methylpyridyl)porphyrinatopalladium(II), PdP(2), was investigated by electronic magnetic circular dichroism (MCD), natural circular dichroism (CD), and optical (UV-visible) absorption spectroscopies. We conclude that four-coordinate, “thick” PdP(2) binds to the exterior of each of poly(A-T)2 and calf thymus DNA (CT DNA) by two distinctly different AT-specific minor and major groove modes, with site 5′TA3′ being favored for both modes. The minor groove mode involves an edge-on orientation of PdP(2), for which porphyrin electric dipole transition moments (edtms) μx (most perturbed direction of the bound porphyrin) and μy (least perturbed direction) have approximate orientation angles of α/β/β′ = ∼ 90°/0°/0° and ∼ 45°/0°/90°, respectively. Major groove binding is by a face-on mode, which results in the porphyrin plane being approximately parallel to the helix axis, such that νx (most perturbed direction) and μy (least perturbed direction) have approximate orientation angles of α/β/β′ = ∼ 45°/180°/90° and ∼ 45°/180°/270°, respectively. The Soret MCD and optical band alterations upon binding (i.e., sign retention of the tetragonal, genuine MCD (+) A-term on becoming the (+) pseudo-A-term of similar amplitude and small DNA-induced optical red (Δλ) and hypochromic (H) shifts) are all consistent with exterior binding perturbations of the porphyrin's pπ MOs (1a1u3a2u 4eg) by the A and T bases of each polymer being weaker than caused by intercalation. Furthermore, that the (+) A-term of PdP(2) retains the (+) sign upon binding informs that the 4eg splitting, or ΔLUMO, is less than the energy separation |1a1u-3a2u|, or ΔHOMO. For the third system, PdP(2)/poly(G-C)2, the B0 CD spectrum has two extremely weak (+) and (−) CD bands at higher and lower energy, respectively, indicating that weak outside binding (wob) interactions are taking place between the cationic porphyrin and the electron-rich phosphate backbone of this rigid polymer. The composite of our CD, MCD, and optical data are suggestive of a face-on mode at the GC major groove. Band parameter extraction is performed on the Soret CD and MCD bands of each of the three bound systems, and it is determined that (1) very little spatial rotation of molecular charge is induced during CD excitation and (2) the excited state angular momentum, 〈Lj〉, changes very little upon binding of PdP(2) to each duplex. These findings are also consistent with each PdP(2)/B-DNA interaction not being very strong. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 341–352, 1998

Active Site Analysis of P450 Enzymes: Comparative Magnetic Circular Dichroism Spectroscopy

Recent structural studies indicate that the substrate- and O2-binding distal pocket of the P450 enzymes are not identical. Thus, P450terp (CYP108) from the α-terpineol-metabolizingPseudomonaddiffers from P450cam (CYP-101) (C. A. Hasemannet al., J. Mol. Biol.236, 1169, 1994). In contrast, the distal pockets of P450terp and P450BMP (CYP102 heme domain;Bacillus megaterium) are more closely similar, including novel hydrogen-bonding interactions between the distal H2O ligand and the I helix (C. A. Hasemannet al., Structure,3, 41–62, 1995). To evaluate the significance of these differences, we have compared solution magnetic circular dichroism (MCD) spectra of P450terp with spectra of other P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase. Spectra of native P450terp are more similar to those of P450BMP and those of mammalian P450LM-2 than to those of P450cam. Upon substrate-binding, the MCD spectra of ferric P450terp and all other thiolate-ligated heme systems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or other 5-coordinate ferric heme systems. This intense negative MCD feature thus appears diagnostic for cysteinate-linked ferric hemes. In the case of ferrous P450s, the intensity of the Soret-region MCD trough varies between substrate-bound and substrate-free enzymes (despite the fact that the substrate is NOT in direct contact with the heme moiety). A novel finding of particular interest is the clear spectral shifts of the Soret MCD band between the substrate-bound and substrate-free forms of ferrous-CO-P450terp. No such observation has been made previously. Furthermore, the band positions for BOTH types of P450terp are red-shifted from known bands of ferrous-CO-P50cam. These data thus indicate a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on CO-binding rates and equilibrium constants. Comparative analysis of the spectral properties of P450terp with MCD spectra of other P450 enzymes, as well as with chloroperoxidase and NO synthase, demonstrates both the expected similarities and the significant differences that reflect active-site structural features. The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous-CO-P450. Furthermore, testable structural predictions for P450-BM-1 and for the novel NO synthase enzyme (neither of which has been crystallized to date) are made herein. This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes.

Low-temperature magnetic circular dichroism investigation of the active site of chloroperoxidase

Magnetic circular dichroism (MCD) spectra for near UV and visible spectral regions of chemically reduced chloroperoxidase from Caldariomyces fumago have been recorded at temperatures from near 293 to 2.15 K. The spectra of reduced chloroperoxidase at 4.2 K were compared with those of photolysis products of its carbon monoxide complexes. Obtained results give evidence for high rigidity of the active site in chloroperoxidase and strongly suggest that thiolate is a protein-derived ligand of the heme iron in the reduced enzyme. The unusual high-spin ferrohemoproteins temperature dependence of the Soret MCD closely resembles that of the substrate-bound cytochrome P-450 cam.

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.

Formation of high spin cytochrome a in isolated cytochrome c oxidase.

The effect of temperature on the electronic structure of cytochromes a and a3 has been determined at two different concentrations of the solubilizing detergent, N-dodecyl beta-D-maltoside. In each case, an incubation temperature of 40 degrees C resulted in a 20-30% increase in the 430-nm conformer of cytochrome a3 when compared with a 20 degrees C control, but the higher detergent concentration led to additional formation of a new charge transfer band at 630 nm. Induction of this new feature was correlated with a loss of alpha-band intensity, a blue shift in the Soret absorbance, and a decrease in the Soret magnetic circular dichroism. The absorbance changes were associated with isosbestic wavelengths at 613, 581, 563, 453, and 416 nm, indicating a two-state electronic conversion. The loss of magnetic circular dichroism intensity and the lack of an isosbestic point between 630 and 655 nm were consistent with a selective effect on the electronic structure of ferricytochrome a. Comparison of these spectral changes with the model heme a studies of Carter and Palmer (Carter, K., and Palmer, G. (1982) J. Biol. Chem. 257, 13507-13514) provides evidence for a low to high spin transition in cytochrome a in which one of the axial Fe-N bonds is either broken or sterically strained. This selective effect on cytochrome a and the associated thermal resistance of the a3-CuB domain are discussed in terms of the 4-helix bundle motif that has been recently proposed for the catalytic site of cytochrome c oxidase.

Spectroscopic studies on lacquer peroxidase from Rhus vernicifera and its derivatives

In order to probe the active site of lacquer peroxidase (LAP) from Rhus vernicifera, electronic absorption and magnetic circular dichroism (MCD) studies of the enzyme with and without various exogenous ligands (NO, CO, and CN −) have been carried out at room temperature. The absorption and MCD spectra of native LAP are quite similar to those of horseradish peroxidase (HRP), indicating that the enzyme has a heme with the fifth and the sixth coordination sites occupied by histidine imidazole and an exogenously exchangeable ligand (H 2O), respectively. Moreover, the superhyperfine structure of the electron paramagnetic resonance (EPR) signal of NO-ferrous LAP provides strong support for the fifth imidazole ligand. A comparison of the Soret MCD band of native LAP with that of Hg(II)-treated LAP demonstrated that an equilibrium mixture of high-spin heme (major species) and low-spin heme (minor species) is present in the enzyme at room temperature. All of the derivatives of NO, CO, and CN − exhibit electronic and MCD spectra of low-spin complexes; similar spectral features are found for the corresponding derivatives of HRP and myoglobin (Mb). The treatment of LAP with H 2O 2 gives rise to stable compound II at room temperature, while compound I is too unstable to be spectroscopically observed.

Effects of cyanogen bromide modification of the distal histidine on the spectroscopic and ligand binding properties of myoglobin: magnetic circular dichroism spectroscopy as a probe of distal water ligation in ferric high-spin histidine-bound heme proteins

Magnetic circular dichroism (MCD) spectroscopy has been utilized to characterize the change in coordination structure in native ferric sperm whale myoglobin upon cyanogen bromide-modification. Comparison of the MCD properties of the ferric high-spin state of cyanogen bromide-modified myoglobin (BrCN-Mb) with those of native ferric horseradish peroxidase and Aplysia myoglobin suggests that ferric BrCN-Mb is a potential MCD model for the pentacoordinate state of ferric high-spin histidine-ligated heme proteins. These five-coordinate heme proteins afford a relatively weak and unsymmetric signal in the Soret region of the MCD spectrum. In contrast, native ferric myoglobin and the benzohydroxamic acid adduct of ferric horseradish peroxidase show a strong and symmetric derivative-shaped Soret MCD signal which is indicative of hexacoordination with water and histidine axial ligands. Therefore it seems that MCD spectroscopy could be used to probe the presence of water ligated to the distal side of ferric high-spin heme proteins. The MCD spectra of the ferric-azide, ferrous-deoxy and ferrous-CO forms of BrCN-Mb have also been measured and compared to those of analogous native myoglobin complexes. The present MCD study has been extended to include new ligands, NO, thiocyanate and cyanate, which bind to ferric BrCN-Mb. With exogenous ligands such as CO, NO and thiocyanate, the coordination structures of the BrCN-Mb complexes are similar to those of the respective native myoglobin adducts. In the case of ferrous-deoxy and ferric-cyanate BrCN-Mb, however, the altered MCD spectra (and EPR for the latter) reveal changes in electronic structure which likely correlate with alterations of the coordination environment of these BrCN-Mb derivatives. Data are also presented which support the proposed tetrazole-bound structure for azide-treated BrCN-Mb (Hori, H., Fujii, M., Shiro, Y., Iizuka, T., Adachi, S. and Morishima, I. (1989) J. Biol. Chem. 264, 5715-5719) and the inability of the distal histidine of BrCN-Mb to stabilize the ferric ligand-bound state.

Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents α + and β chains for α 2 + β 2, α and β + chains for α 2 β 2 +: + denotes ferric heme) were measured in the Soret region for F −, H 2O, N 3 − and CN − derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F − and H 2O of α 2 + β 2 displayed a blue shift in the peak position around 430 nm and those of α 2 β 2 + an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy β chains of α 2 + β 2 and deoxy α chains of αβ 2 +, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different frm the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the α 1 β 1 subunit contact.

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).

Magnetic circular dichroism of myoglobin-thiolate complexes

Various complexes of myoglobin (Mb) with thiolate were studied by use of magnetic circular dichroism (MCD) spectroscopy. 1. 1.|MetMb-ethyl, n-propyl and isopropylmercaptan complexes offered MCD spectra similar to that of cytochrome P-450( P-450) with respect to shape and intensity ratio of Soret MCD to Q 0–0 MCD. The MCD spectra did not show any pH dependence. The complexes reduced by sodium dithionite exhibited the MCD spectrum of deoxyMb, indicative of release of thiolate anion from the heme iron. 2. 2.|Cysteine and cysteine methyl ester coordinated to the heme iron at pH 9.18, but not at pH 6.86 and 11.45. The complex formed at pH 9.18 gave an MCD spectrum similar to that of P-450, and an MCD spectrum of deoxy Mb on reduction with sodium dithionite. 3. 3.|The 2-mercaptoethanol complex exhibited three A terms associated with the Q 0–0, Q 0–1, and Soret transitions at pH 6.86 similar to those of Fe(II) cytochrome c, which indicates that Mb was reduced by this reagent at pH 6.86. At pH 9.18 2-mercaptoethanol gave an MCD spectrum similar to that of alkyl mercaptan just after the addition. With time it changed into deoxy Mb through some intermediate of reduced Mb-thiolate complex. At pH 11.45 2-mercaptoethanol formed complex which exhibited an MCD spectrum similar to those of other alkylmercaptans. 4. 4.|Sodium sulfide gave an MCD spectrum which resembled that of the normal thiol Mb complex just after addition at pH 6.86. The complex was gradually reduced to give a 610 nm trough in addition to the MCD of deoxyMb. The Mb-sulfur complex formed at pH 9.18 was gradually reduced to give an MCD spectrum which was fairly different from that of deoxyMb. A similar MCD spectrum was observed at pH 11.45 just after the addition of Na 2S. These results were considered to suggest the saturation of one of the conjugated double bonds of the porphyrin by sulfur.

Magnetic circular dichroism studies of pyridine-heme complexes in aqueous media

The magnetic circular dichroism (MCD) spectra for Fe 3+ and Fe 2+ protoporphyrins (hemin and heme) with and without pyridine have revealed the sensitivity of the electronic structures of the complexes formed to the solvent system: (1) The hemin in an aqueous ethylene glycol solution gave the practically same Q and Soret MCD as that in an alkaline aqueous solution, indicating that there is no fundamental differences in the electronic states between the monomeric and the dimeric hemins, and that they are both in high spin states. (2) The hemin-pyridine complex in an aqueous solution showed an MCD essentially similar to that for the hemin described above in (1), indicating that this complex is in the high spin state. (3) The addition of pyridine to the monomeric hemin in an aqueous ethylene glycol solution induced the typical spin change from high to low like myoglobin derivatives. (4) The heme in an aqueous solution showed the MCD fairly different from that in an aqueous ethylene glycol solution, the latter showing the MCD of monomeric heme of deoxy-myoglobin. This indicates the existence of the heme-heme interaction in the aggregated or dimeric high-spin heme. (5) The heme-pyridine complexes showed the MCD characteristic of Fe 2+ low-spin complex irrespective of the solvent system.