Magnetic circular dichroism of adenine, hypoxanthine, and guanosine 5'-diphosphate to 180 nm

The magnetic circular dichroism (MCD) of adenine, hypoxanthine, and guanosine 5′-diphosphate reveals that, for each species, the uv-absorption band near 200 nm is composed of at least two electronic transitions. The theory of MCD shows that the dipoles of these transitions are not parallel to one another. The transitions are assigned within the framework of current theories of the electronic structure of the purines. Knowledge of the presence of more than one transition within an absorption envelope is important in interpreting the corresponding natural CD of DNA and RNA.     

An evaluation of methods for sampling macrophyte maximum colonisation depth in Loch Leven, Scotland

The maximum colonisation depth (MCD) of macrophytes in lakes is considered a robust ecological indicator of system health. The accuracy of five boat-based survey methods (double-headed rake, Ekman grab, bathyscope, hydroacoustics, and underwater photography) was evaluated along depth-transects within a shallow eutrophic loch (Loch Leven, Scotland, UK) in 2006. Data from an earlier field campaign conducted in 1993 (33 transects) were used to assess sample frequency and error associated with the use of the double-headed rake method. The double-headed rake consistently sampled more species than the Ekman grab or bathyscope, probably as a result of its ability to sample relatively larger areas of the lake bed in comparison to the other methods. The bathyscope method underestimated the MCD in comparison to all other methods. This appeared to be because the turbidity of the water-column restricted the depth of view when using the bathyscope. The double-headed rake and underwater photography methods were in good agreement, with maximum observed MCD estimates of 5.0 m and 4.5 m water depth, respectively (2006). Using the 1993 data (MCD observations taken from 33 selected transects), we estimated that over 5 transects were required to ensure a MCD estimate greater than the 75th percentile from the distribution of all 33 colonisation depths, more than 15 replicate transects were required to ensure an estimate greater than the 90th percentile, and more than 20 replicate transects were required to ensure an estimate greater than the 95th percentile.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

The energy level scheme for the ferryl heme in compound II of the peroxidase-catalase family as determined from analysis of low-temperature magnetic circular dichroism

The expressions for temperature-dependent magnetic circular dichroism (MCD) of the ferryl heme (Fe(4+)Por, S=1), which is a model of an intermediate product of the catalytic cycle of heme enzymes (compound II), have been derived in the framework of a two-term model. Theoretical predictions for the temperature and magnetic field dependence of MCD intensity of the ferryl heme are compared with those of the high-spin and low-spin ferric heme. Analysis of reported MCD spectra of myoglobin peroxide [Foot et al., Biochem. J. 2651 (1989) 515-522] and compound II of horseradish peroxidase [Browett et al., J. Am. Chem. Soc. 110 (1987) 3633-3640] has shown the presence in the samples of approximately 1% of a low-spin ferric component, which, however, should be taken into account in simulating observed temperature dependences of MCD intensity. The values of two adjustable parameters are estimated from the fit of the observed and simulated plots of MCD intensity against the reciprocal of the absolute temperature. One of them, the energy gap between the ground and excited terms, predetermines the axial zero-field splitting. The other parameter is correlated with the energy of splitting of excited quartets arising from either the porphyrin pi-->pi* transition or the spin-allowed charge-transfer transition.     

How mantids gain insight into the new maximum catching distance after each ecdysis

The maximum catching distance (MCD) of which a mantid, Stagmatoptera biocellata, can successfully strike a prey keeps a certain relationship with its foreleg extension in each instar. Two hypotheses aimed at explaining the occurrence of this relationship in spite of the sudden growth in dimensions that takes place after each ecdysis are discussed. The assumption that structures involved in depth perception show changes that allow the mantid to estimate correctly the MCD is tested. This ‘automatic adaptation’ hypothesis receives strong support from the results. They indicate that throughout postembryonic development the MCD has a constant proportionality with three head dimensions, i.e. the head breadth, the ocular globe breadth, and the ocular prominence. These modifications during the saltatory growth cause a change in the optical triangulation system that can, by itself, explain the perfect estimate of the distance within which a strike is a worth-while effort, without resorting to a learning hypothesis.     

Magnetic circular dichroism study on synthetic polynucleotides, bacteriophage structure, and DNA's

The MCD (magnetic circular dichroism) spectra of Ap, ApA, ApApA, poly A, Up, UpU, poly U and double-stranded poly A:U alternating copoly A–U and alternating deoxyribopoly A–T were measured with a Cary 61 spectropolarimeter fitted with a Varian superconducting magnet at a field strength of 50 Kgauss. The MCD spectra of T2 and T5 DNA at various stages of heal denaturation were measured as a function of hyperchromicity of the sample. MCD spectra of the intact and degraded T2 and T5 phages were used to study the degree of alteration of the DNA inside the phages versus the DNA in vitro. The results for the adenine polymers show that the main MCD bands, B_2u(271 nm), B_1u(252 nm), and E_1u(212 nm), show a decrease in specific magnitude as the length of the polymer is increased, reflecting the degree of stacking of the polymer. In contrast, the uridine series of polymers shows little change of the MCD bands, indicating that there is little interaction between the bases regardless of the length of the polymers. The MCD spectra of poly A:U, alternating poly r(A–U): (A–U), and alternating poly d(A–T):(A–T) show significant differences among themselves in the magnitude of the B_2u band and when compared with the sum of the spectrum for the poly A plus poly U. This may indicate the selective effect of hydrogen bonding on the B_2u band. Alternatively, the difference may be due to the absence of an n → π* transition in the double-stranded polymer. Measurements of denatured T2 and To DNA's show increases in all MCD bands. The T2 DNA internally packed in phage shows an increase of the B_2u and E_1ubands, the B_2u remaining unchanged. The internal T5 DNA shows an increase of the B_1u band only. Thus, the internal DNA structure is altered in a manner quite different from a simple denaturation caused by hydrogen bond breaking. Furthermore, different MCD bands indicate that different modes of DNA packing exist for T2 and T5 phages.     

Procyanidins can interact with Caco-2 cell membrane lipid rafts: Involvement of cholesterol

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol–Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex–cholesterol bondings. Procyanidin–lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.     

Amelioration of diet-induced nonalcoholic steatohepatitis in rats by Mn-salen complexes via reduction of oxidative stress

Background

Nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), is characterized by steatosis (accumulation of triacylglycerols within hepatocytes) along with inflammation and ballooning degeneration. It has been suggested that oxidative stress may play an important role in the progress of NAFLD to NASH. The aim of present study was to determine whether antioxidant supplementations using EUK-8, EUK-134 and vitamin C could improve the biochemical and histological abnormalities associated with diet-induced NASH in rats.

Methods

NASH was induced in male N-Mary rats by feeding a methionine - choline deficient (MCD) diet. The rats were fed either normal chow or MCD diet for 10 weeks. After NASH development, the MCD-fed rats were randomly divided into four groups of six: the NASH group that received MCD diet, the EUK-8 group which was fed MCD diet plus EUK-8, the EUK-134 group which was fed MCD diet plus EUK-134 and the vitamin C group which received MCD diet plus vitamin C. EUK-8, EUK-134 and vitamin C (30 mg/kg body weight/day) were administered by gavage for eight weeks.

Results

Treatment of MCD-fed rats with salens reduced the sera aminotransferases, cholesterol, low density lipoprotein contents, the extent of lipid peroxidation and protein carbonylation whereas the HDL-C cholesterol levels were significantly increased. In addition, EUK-8 and EUK-134 improved steatosis, ballooning degeneration and inflammation in liver of MCD-fed rats.

Conclusion

Antioxidant (EUK-8, EUK-134 and vitamin C) supplementation reduces NASH-induced biochemical and histological abnormalities, pointing out that antioxidant strategy could be beneficial in treatment of NASH.     

Magnetic circular dichroism studies of myoglobin,hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q ₀₀- and Q_v-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q ₀₀-band to the A-term in the Q _v-band. The evidences are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the hemoglobin-like MCD, while the alkaline ferroperoxidase is characterized by the myoglobin-like MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoproteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-ion charge-transfer electronic transition which may be assigned as b() 3d. This charge-transfer band is strongly overlapped with usual B( - *) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Application of C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

The liquid chromatography–multiple reaction monitoring–tandem mass spectrometry (LC–MRM–MS/MS) method using ¹³C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting ¹³C-labeled peptides in blood sample with a C₁₈ cartridge, the extract was applied to a ¹³C monoisotopic transition LC–MRM–MS/MS system with d-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5–100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The ¹³C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three ¹³C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each ¹³C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC–MRM–MS/MS method.     

Spectroscopic studies of nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: nature of the iron-sulfur clusters

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum utilizes three types of Fe-S clusters to catalyze the reversible oxidation of CO to CO(2): a novel [Ni4Fe5S] active site (C cluster) and two distinct [4Fe4S] electron-transfer sites (B and D clusters). While recent X-ray data show the geometric arrangement of the five metal centers at the C cluster, electronic structures of the various [Ni4Fe5S] oxidation states remain ambiguous. These studies report magnetic circular dichroism (MCD), variable temperature, variable field MCD (VTVH MCD), and resonance Raman (rR) spectroscopic properties of the Fe-S clusters contained in Ni-deficient CODH. Essentially homogeneous sample preparations aided in the resolution of the reduced [4Fe4S](1+) (S = (1)/(2)) B cluster and the reduced Ni-deficient C cluster (denoted C, S > (1)/(2)) by MCD. The three Fe atoms derived from the [Ni3Fe4S] cubane component appear to dominate the reduced C cluster MCD spectrum, while the presence of a fourth Fe center can be inferred from the ground state spin. The same underlying MCD features present in Ni-deficient CODH spectra are also observed for Ni-containing CODH, suggesting that both proteins contain the same C cluster Fe-S component. Overlooked in all spectroscopic studies to date, the D cluster was confirmed by rR to be a typical [4Fe4S] site with cysteinyl coordination. Together, MCD and rR data show that the D cluster remains in the oxidized [4Fe4S](2+) (S = 0) state at potentials > or = -530 mV (versus SHE), thus exhibiting an unusually low redox potential for a standard [4Fe4S](2+/1+) electron-transfer site.     

A light-scattering pressure-jump kinetics apparatus.

We have developed an optical sample cell made of stainless steel and fitted with three quartz ultracentrifuge windows in standard holders, to follow the kinetics of macromolecular reactions by the pressure-jump technique. Photomultiplier response to transmitted white light is continuously subtracted from photomultiplier response to white light scattered at 90°C, the difference being displayed by an oscilloscope. The pressure is simultaneously monitored by a quartz pressure sensor in mechanical contact with the sample. Pressurization is accomplished by leading in gas from a commercial cylinder, as originally described by Ljunggren and Lamm, but the pressurization time has been reduced by a factor of 25, to 2 millisec, by valving off a fixed volume of helium and introducing it into the sample cell through a high-speed solenoid valve. Determinations may be repeated at will on a single sample, of total volume under 2 ml. This light-scattering pressure-jump apparatus has been used to observe the kinetics of a number of macromolecular interactions and to determine rate constants for the ribosome-subunit interaction of Escherichia coli.     

Visible region MCD and MLD spectra of nitrosylferrohemoglobin and oxyhemoglobin

Magnetic circular dichroism (MCD) and magnetic linear dichroism (MLD) spectroscopies at various applied magnetic fields (0-6T) and temperatures (2.0-31K) have been used to investigate the electronic properties of the visible (Q(0-0), or alpha band) region of oxy- and nitrosylferrohemoglobin (HbNO). OxyHb, a d(6) (S=0) diamagnet, exhibits the expected pseudo-first derivative MCD and pseudo-second derivative MLD temperature-independent features centered at 574nm. HbNO, a d(7) (S=1/2) paramagnet, also exhibits a temperature-independent pseudo-first derivative MCD spectrum, but centered at 571nm. So far as we are aware, this behavior is unprecedented in the MCD spectra of paramagetic iron-porphyrins, which are expected to be dominated by temperature-dependent C(0) terms. The HbNO MCD spectrum does, however, demonstrate limited field-dependent saturation magnetization behavior and the MLD spectrum is currently below the detection limit. In addition, an MCD signal from reoxygenated venous blood is reported and compared with MCD signals from oxy- and HbNO derivatives. Finally, a combination of MCD and MLD spectroscopies has been used to estimate the orbital angular momentum (M(L)) value of the alpha band excited state of oxyHb as 4.2 (+/-0.7).     

Key cofactors of Photosystem II cores from four organisms identified by 1.7-K absorption, CD and MCD

Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98–103]. Electrochromic shifts resulting from Q_A reduction by 1.7-K illumination were recorded, and the Q_x and Q_y absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Q_x transition is ∼3 nm (100 cm⁻¹) to higher energy in cyanobacteria than in the plants. The predominant Q_y shift appears in the range 683–686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Q_y region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.     

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.     

Recent applications of MCD spectroscopy to metalloenzymes

Magnetic circular dichroism (MCD) continues to be a powerful probe of metalloenzyme electronic and geometric structure, in addition to playing a major role in the determination of heme enzyme coordination geometries. Excited state electronic structure contributions to enzyme activity have been gleaned from C-term MCD studies, which are usually interpreted in the context of other spectroscopies, including electronic absorption and resonance Raman. The recent development of sophisticated methods for the analysis of variable-temperature, variable-field MCD have allowed the ground states of metalloenzyme active sites to be studied in detail, providing information on the electronic and geometric structure of the site. This has been especially informative for non-heme iron enzymes. In the past two years X-ray MCD has been shown to be a promising technique for the study of metalloenzymes.     

Structured near-infrared Magnetic Circular Dichroism spectra of the Mn4CaO5 cluster of PSII in T. vulcanus are dominated by Mn(IV) d-d ‘spin-flip’ transitions

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn₄CaO₅. VTVH MCD spectra taken in the S₂ state provide a clear g = 2, S = 1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749 nm, negative (?) at 773 nm and (+) at 808 nm are assigned as ⁴A → ²E spin-flips within the d³ configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S₃ exhibits a more intense (?) MCD peak at 764 nm and has a stronger MCD saturation characteristic. This S₃ MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S₂ EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.