Soret Transition Research Articles

Resonance Raman scattering as a probe of electron—nuclear coupling: comparison of the relative intensity and absolute cross-section transform methods

We present the results of a resonance Raman study of the overtone and combination bands of ferrocytochrome c. These bands are weak, but they can still be resolved in rosonance with the Soret transition and compared to the fundamental intensities in order to extract the electron—nuclear coupling without the use of absolute scattering cross-sections. As a result, we can test the absolute cross-section transform method, which determines the coupling by scaling the measured light scattering intensities to a value predicted by a Kramers-Kronig transform. The comparison of the two methods is encouraging.

Structure of the primary electron donor in photosystem I: a resonance Raman study

Low-temperature resonance Raman (RR) spectra have been obtained at resonance with the Soret transition of chlorophyll a in photosystem I particles containing large amounts either of the triplet state of P700 or of its radical cation state. Subtracting these spectra from those of resting reaction centers yielded RR spectra of P700 in its neutral, ground state. These spectra arise from two distinct chlorophyll a molecules differing by the strengths of the bonding interactions assumed by their keto carbonyl groups, the stretching frequencies of which are found at 1655 and 1675 cm-1. The present results rule out previous hypotheses that P700 might have consisted of a single, chemically modified chlorophyll a molecule. Neither of the bonding interactions assumed by the keto carbonyls of the P700 chlorophylls most probably involves chlorophyll-chlorophyll bridging through water molecules, as surmised in the so-called special pair models, but likely consists of H bonds with distinct protein sites. The magnesium atoms of the two P700 chlorophylls are 5-coordinated. Hence, the structural model of P700 provided by the present data is qualitatively the same, in terms of bonding interactions, as that currently accepted for the bacterial primary donor.

Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement.

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.

Resonance Raman studies of oriented chromophores: Metmyoglobin single crystals

We report Soret enhanced resonance Raman studies of single crystals of metmyoglobin. Differing scattering intensities of the 345 cm−1 mode in solution and crystalline forms of the protein indicate a structural perturbation of the heme upon crystallization, possibly involving a propionate substituent of the heme. The immobilization of the heme in the crystal also allows observation of low-quantum-yield photoreduction of the heme that appears to be driven by Soret band absorption. We present experimental investigations of the orientational dependence of the intensity and polarization of several strong heme Raman modes along with calculations that explore the effects of vibrational mode symmetry and of differential coupling to x-, y-, and z-polarized electronic transitions. Orientation studies provide evidence that the vinyl C=C stretching band at 1622 cm−1 is a superposition of two independent modes that couple selectively to the x- and y-polarized Soret transitions. We also demonstrate how modes of nominal B1g and B2g symmetry can be distinguished by crystal studies. Modes that couple preferentially to charge transfer transitions polarized perpendicular to the heme plane are shown to be easily distinguishable from the majority of heme modes, which couple to the in-plane polarized Soret transition; we observe no modes of the former type in metmyoglobin. Finally, we present calculations showing that single-crystal polarization studies of resonance Raman scattering as a function of excitation frequency should be sensitive to the splitting of the nearly xy-degenerate Soret band as well as to the orientation of the x- and y-polarized transitions.

Metalmetal bonds extended over a porphyrin ring. II. Resonance raman and far-infrared spectra of metalloporphyrins containing InM bonds

The low-frequency resonance Raman (RR) and far-IR spectra of (TPP)InM(CO) 3Cp (M = Mo and W) are reported. The ν(InM) vibrations were located at ∼ 160 and 147 cm −1, respectively, for the Mo and W compounds. The RR excitation profiles were used to distinguish ν(InM) from TPP vibrations which dominate the low-frequency spectra and to demonstrate that the split component of the Soret transition near 460 nm contains some σ(InM)σ*(InM) character.

Resonance Raman studies of sterically hindered cyanomet "strapped" hemes. Effects of ligand distortion and base tension on iron-carbon bond

We report resonance Raman studies of the iron-carbon bond stretching vibrations, nu(Fe-CN), in sterically hindered and unhindered heme (FeIII)-CN- complexes. The sterically hindred "strapped hemes" are equipped with a covalently linked 13-, 14-, or 15-atom hydrocarbon chain across one face of the heme; these are called FeSP-13, FeSP-14, and FeSP-15, respectively. These straps would presumably exert a sideway shearing strain to force the linear ligands (e.g., CN- and CO) to be tilted and/or bent. The shorter the chain length, the weaker the ligand binding affinity because of a greater steric hindrance. This study reveals that the nu(Fe-CN) frequency decreases as the chain length is decreased, in contrast with the CO complexes, where the nu(Fe-CO) frequency increases as the chain length is decreased. For the heme-CN- complexes (with N-methylimidazole as a base), the nu(Fe-CN) frequencies are: heme 5 (unhindered), 451 cm-1; FeSP-15, 447 cm-1; FeSP-14, 447 cm-1; FeSP-13, 445 cm-1. For the heme-CO complexes (with N-methylimidazole as a base), the nu(Fe-CO) frequencies are: heme 5, 495 cm-1; FeSP-15, 509 cm-1; FeSP-14, 512 cm-1; FeSP-13, 514 cm-1 (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang, 1983, Biochemistry, 22:4534-4540). We have also studied the cyanide complexes with three different bases (pyridine, N-methylimidazole and 1,2-dimethylimidazole), and found that the trans-effect of cyanide complex is different from that of CO complexes. The tension on Fe"'-base bond weakens the Fe"'-CN- bond, whereas the tension on Fe"-base bond strengthens the Fe"-CO bond. The origin of these differences may be attributed to different extents of the d7r(Fe)- wr*(ligand) back bonding between the CN- and CO heme complexes. The Fe-C-N bending vibrations in these cyanomet strapped hemes are not resonance-enhanced, although this bending mode has been detected at -410 cm-' via Soret excitation in cyanometinsect hemoglobins. It is suggested that the orientation of the tilted Fe-C-N unit may be important in determining the overlap between CN and porphyrin 7r* orbitals, which provide coupling of the Fe-C-N bending mode with the resonant Soret (r-7r*) transition.

Observation of induced cotton effects for magnesium porphyrin-L-histidine species

Complexes of magnesium protoporphyrin and magnesium mesoporphyrin with L-histidine have been prepared in solution and studied by circular dichroism and optical rotatory dispersion spectroscopy. Large induced Cotton effects are observed, in particular for the Soret transitions. These are interpreted in terms of the formation of six-coordinate magnesium porphyrin-(L-histidine) 2 species where the asymmetry of the L-histidine groups is coupled to the magnesium porphyrin electronic transitions. The major, five-coordinate components of these solutions do not produce Cotton effects. These results, when compared with data for corresponding protein complexes, lend support for the coupled oscillator description of the origin of optical activity in hemoproteins. The observation of Cotton effects for porphyrin complexes of the type reported here may enable the existence of six-coordinate species to be more clearly established in instances where the major equilibrium product is a five-coordinate species.

Griffith model bonding in dioxygen complexes of manganese porphyrins

The binding of O/sub 2/ to Mn(II) porphyrins greatly alters their optical and EPR spectra. The optical spectra of these pentacoordinate complexes are transformed from the normal to hyper type with a split Soret band typical of Mn(III) porphyrins. EPR spectra indicate a spin change from S = 5/2 to S = 3/2, and /sup 17/O substitution reveals little unpaired spin density on the O/sub 2/. Contradictory interpretations of the electronic and geometric structures of these complexes have recently been advanced: analysis of the EPR data supports a d/sup 3/ Mn/sup IV/O/sub 2//sup 2 -/ with O/sub 2/ bound in the Griffith mode (edge-on, parallel to the porphyrin plane) whereas ab initio calculations favor a d/sup 2/..pi..* Mn/sup III/O/sub 2//sup -/ with O/sub 2/ bound in the Pauling mode (end-on, bent). Charge iterative extended Hueckel calculations on both Griffith and Pauling models of oxymanganese porphine are reported here in which the O-O, Mn-O, and out-of-plane Mn distances and the O-O orientation above the porphinato plane were varied. For all the calculations, the porphine ring as well as the metal transfers considerable charge density onto the O/sub 2/, suggesting that a formal charge description of these complexes solely in terms ofmore » the O/sub 2/Mn core is an oversimplification. The resultant wave functions were used to calculate /sup 55/Mn and /sup 17/O hyperfine splittings. Griffith models with three unpaired electrons occupying orbitals of predominantly d character give excellent agreement with the observed /sup 55/Mn and /sup 17/O values. The optical hyperspectra can be ascribed to porphine ..pi.. ..-->.. d/sub x/,O/sub 2/..pi..* charge-transfer transitions that mix with the porphine ..pi.. ..-->.. ..pi..* Soret transitions. All the experimental results can be explained in terms of a d/sup 3/ configuration and Griffith binding of the dioxygen but not Pauling binding.« less

Femtosecond relaxation of an iron porphyrin observed with polarization spectroscopy in a three-level system

An experimental and theoretical study is presented of the nonlinear response of a dilute solution being pumped by two laser fields having different frequencies omega(1) and omega(2) that are both in resonance with electronic transitions of the solute. Experimental results were obtained for the dispersion of the third-order susceptibility chi((3))(omega(2))(2) for a dilute solution of iron(III) tetraphenylporphyrin chloride in chloroform with a fixed intense field (omega(1)) resonant with the Q(o) transition and a tunable weaker field (omega(2)) resonant with the Soret transition. The observed Lorentzian dispersion is slightly narrower than the linear absorption spectrum. The theoretical model for the third-order polarization incorporates three levels coupled to two electromagnetic fields and a population reservoir. The equations of motion for the density operator were solved in third order for the appropriate Fourier component. The resulting dispersion curve depends on both total dephasing and population decay rates, thereby demonstrating a general method for the determination of subpicosecond relaxation processes in systems to which the model is applicable. Theoretical fits to the results for iron(III) tetraphenylporphyrin chloride imply that the Soret transition is essentially homogeneously broadened with a total dephasing width of 900 +/- 200 cm(-1), and it is argued that the population decay time in this case is approximately 3.5 fsec. The broad linear absorption spectrum of the Soret band is interpreted as resulting from intramolecular perturbations between the Soret and lower energy excited states of iron(III) tetraphenylporphyrin chloride.

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes

Circular dichroism (CD) spectra were measured for cytochromes P-450 ( P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in α-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45–50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250–310 nm) and weaker negative CD spectra associated with Soret transitions for PB P-448 ([ φ] = 50 000) and MC P-448 ([ φ] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.

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.

Optical rotatory dispersion and circular dichroism of cytochrome oxidase.

Optical rotatory dispersion of the oxidized and the reduced cytochrome oxidase has been measured from 196 to 550 nm. The experimental data have been analyzed in order to separate the extrinsic cotton effects from the rotations of the protein backbone. From the resolution, two cotton effects of similar sign are obtained in the Soret region. For the reduced enzyme, the cotton effect centered at 446.5 nm shows a molar amplitude of 192 k‐degrees with a band width of 5.3 nm and rotational strength of 0.25 Debye magneton; the second cotton effect centered at 444 nm shows a molar amplitude of 45 k‐degrees with a band width of 12.9 nm and rotational strength of 0.14 Debye magneton. Upon oxidation, the centers of these cotton effects shift to 432 and 423.7 nm. The amplitudes become 114 and 122 k‐degrees with band widths of 6.1 and 11.2 nm and rotational strengths of 0.18 and 0.35 Debye magneton, respectively. In addition, a less defined third cotton effect centered at 376 nm appears with an amplitude of 28 k‐degrees, band width of 18 nm, and rotational strength of – 0.15 Debye magneton. The circular dichroic curves constructed from the optical rotatory dispersion data by the Kronig‐Kramers transform are in general agreement with the experimentally determined spectra.The backbone rotations are analyzed and expressed in the terms of Shechter‐Blout's parameters. The effective helicity thus computed is 50% for the reduced and 44.5% for the oxidized form.The sources of the Soret cotton effects are discussed along with the possibilities of the components present (cytochromes a and a3 and copper), the intrinsic property of the Soret transition, possible splitting of the Soret band, interactions with the protein helix, and heme‐heme and heme‐copper couplings. The fact that the optical rotatory dispersion and circular dichroism behavior is dependent upon the oxidation state of cytochrome oxidase could arise either from an altered conformation of the protein backbone and/or from an altered interaction with the heme group. Regardless of the possibilities, the geometrical conformation of cytochrome oxidase and the electronic relation between the prosthetic group and the apoenzyme is dependent upon the oxidation state of the enzyme.

Conformation of cytochromes: IV. Conformational homology of functionally homologous cytochromes c

The conformational homology of functionally homologous cytochromes c from horse, beef, tuna, turkey, pigeon and moth has been investigated by comparison of the circular dichroism (CD) spectra of each preparation in both oxidation states of iron and in the wavelength region 600-185 mμ at pH 6.8. In the oxidized state all preparations exhibit similar dichroic features with only small variations in the relative magnitudes of various extrema, except for the aromatic circular dichroism curve for tuna cytochrome c. The preparations in the reduced state of iron also exhibit comparable dichroic spectra, both in the region of the Soret transition and the intrinsic absorption region. The CD patterns for the reduced cytochromes in the α-β absorption region, however, exhibit deviations suggestive of small but distinct differences in the electronic asymmetry of the prosthetic groups, especially of the porphyrin moiety, possibly because of differences in the electronically coupled chromophores. A gross conformational homology among various mammalian-type cytochromes c has, therefore, been suggested. The dichroic differences in the case of tuna cytochrome c have been imputed to be a reflection of an unusually asymmetric environment of the residue at position 33 of the polypeptide chain. The intrinsic CD curves above 200 mμ for all the preparations in both valence states of heme iron exhibit features characteristic of the presence of α-helical structures in the molecule. Below 200 mμ, however, the dichroic curves exhibit a single positive maximum at about 194 mμ and a definite minimum at 190 mμ. The former feature is usually associated with systems containing β-structure. The presence of a minimum at about 190 mμ, when taken in conjunction with the characterizing features for α-helical models (a large positive maximum at this wavelength), indicates the absence of α-helically organized structure in the molecule. An analysis of the conformation of the protein moiety in terms of proportions of reference model conformations (α-helix, β-structure and random coil) and based on the recently developed isodichroic method 24 confirms the low α-helical content of various cytochromes and further demonstrates the gross polypeptide conformational homology for various functionally homologous preparations.