Temperature Magnetic Circular Dichroism Spectra Research Articles

Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures

Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.

Resonance Raman and magnetic circular dichroism studies of reduced [2Fe-2S] proteins.

The structural and electronic properties of the [2Fe-2S] clusters in reduced putidaredoxin, Spinacea oleracea ferredoxin, and Clostridium pasteurianum [2Fe-2S] ferredoxin have been investigated by resonance Raman and variable temperature magnetic circular dichroism spectroscopies. Both techniques are shown to provide diagnostic fingerprints for identifying [2Fe-2S]+ clusters in more complex multicomponent metalloenzymes. The Fe-S stretching modes of oxidized and reduced putidaredoxin are assigned via 34S and D2O isotope shifts and previous normal mode calculations for adrenodoxin (Han, S., Czernuszewicz, R. S., Kimura, T., Adams, M. W. W., and Spiro, T. G. (1989) J. Am. Chem. Soc. 111, 3505-3511). The close similarity in the resonance Raman spectra of reduced [2Fe-2S] centers, in terms of both the vibrational frequencies and enhancement profiles of the Fe-S stretching modes, permits these assignments to be generalized to all clusters of this type. Modes primarily involving Fe(III)-S(Cys) stretching are identified in all three reduced [2Fe-2S] proteins, and the frequencies are rationalized in terms of the conformation of the cysteine residues ligating the Fe(III) site of the localized valence reduced cluster. D2O isotope shifts indicate few, if any, amide NH-S hydrogen bond interactions involving the cysteines ligating the Fe(III) site. Preliminary resonance Raman excitation profiles suggest assignments for the complex pattern of electronic bands that comprise the low temperature magnetic circular dichroism spectra of the reduced proteins. S----Fe(III) and Fe(II)----S charge transfer, Fe d-d, and Fe(II)----Fe(III) intervalence bands are identified.

Low temperature magnetic circular Dichroism Spectra and magnetization properties of Extracted Heme a3+ Bis-Imidazole : A Model of Cytochrome a in Bovine Cytochrome c Oxidase

Heme a 3+ isolated from bovine cardiac muscle cytochrome oxidase has been converted to the bis-imidazole species and studied by magnetic circular dichroism (MCD) spectroscopy. Spectra have been recorded down to 1.5°K, enabling the MCD magnetization curves to be measured at a number of wavelengths in the visible and near infrared regions. The experimentally determined curves show excellent correlation to a curve using the g-values determined by electron paramagnetic resonance spectroscopy to be g z = 2.96, g y = 2.29, and g x = 1.73. The data show that the bis-imidazole derivative of extracted heme a 3+ is an excellent model of cytochrome a in the enzyme, confirming the presence of two histidine residues in the protein as the fifth and sixth ligands. The spectral features of heme a 3+ bis-imidazole in the near infrared region are consistent with transitions of the porphyrin ( a 1u, a 2u,) to ferric ( e g ) charge transfer type.

Characterization of the Fe-S cluster in aconitase using low temperature magnetic circular dichroism spectroscopy.

Beef heart aconitase has been studied by low temperature magnetic circular dichroism (MCD) spectroscopy in the wavelength region 300 to 1900 nm. Together with parallel electron paramagnetic resonance and activity measurements, these data enable correlations between Fe-S cluster-type and enzymic activity in aconitase. In samples not exposed to extraneous Fe, the Fe-S cluster in aconitase exhibits the characteristic properties of a 3Fe center in both the as isolated and dithionite-reduced states. On the basis of the detailed form of the low temperature MCD spectra, three types of 3Fe center can be distinguished in biological samples. These are typified by the 3Fe centers in aconitase, Desulfovibrio gigas FdII, and Azotobacter crooccocum Fd. In aconitase, maximal enzymic activity is found to be associated with the build-up of [4Fe-4S]2+ clusters in good agreement with the Mössbauer studies of Kent et al. (Kent, T. A., Dreyer, J. L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1096-1100). However, significant catalytic activity (approximately 60%) was obtained by reduction of the 3Fe center with dithionite in the absence of added Fe. The form and intensity of the resultant MCD spectrum are consistent with the majority of the Fe being in the form of reduced 3Fe clusters. The possibility that a reduced 3Fe cluster is capable of promoting partial catalytic activity in aconitase is discussed in light of these results.

Low-temperature magnetic circular dichroism spectra of metallo-proteins

As an optical probe of the metal centres in proteins magnetic circular dichroism (MCD) spectroscopy has two features of value in biochemistry. First, the MCD spectra of paramagnetic centres are temperature dependent increasing in magnitude as the temperature is lowered, whereas those of diamagnetics are temperature independent. A study of the magnetic filed and temperature dependence of the MCD signal down to 1.5 K and up to 5 Tesla enables the state of magnetisation of a paramagnet to be determined. From such a magnetisation curve it is possible to determine the spin (S) of the ground state and in favourable case, the effective ground state g-factors [1]. Thus identification is possible of optical absorption bands which belong to species detectable by EPR spectroscopy. Furthermore, paramagnets which are invariably invisible to EPR spectrocopy, that is, even-electron spin systems or odd-electron spin paramagnets with M s > ± 1 2 as the lowest energy Kramers doublet, can be investigated. Secondly, the MCD spectra of vibrational transitions are orders of magnitude weaker than those of electronic transitions. Hence electronic states in the MCD spectra of metalloproteins can be detected out to wavelengths as long as 2500 nm. This enables metal localised d-d states and metal-ligand charge-transfer (CT) states to be located. The energies of these states can ben sensitive indicators of the coordination environment of a metal centre in a protein. When applied together these two features of MCD spectroscopy make it a powerful tool in unravelling the electronic structures of metal centres especially those in proteins with more than one centre [2]. We describe several examples drawn from our own work of the last few years. A ferromagnetic haem-copper pair in cytochrome c oxidase. The near infrared MCD spectrum of cyanide-inhibited bovine cytochrome c oxidase shows three bands, at 790 nm, 1564 nm and 1946 nm which can be assigned, on the basis of their MCD magnetisation properties, to Cu 2+ A, the EPR detectable copper ion, cytochrome a 3+, the low-spin EPR detectable haem, and cytochrome a 3+ 3 bridged to Cu 2+ B [3]. The latter apparently comprise a pair of metal ions bridged, probably linearly, by CN − and ferromagnetically coupled to give a ground state spin S = 1, with a zero-field splitting of ∼10–20cm −1 leaving a pair of levels, M s = ±1, virtually degenerate and lower in energy that the M s = 0 component [4]. An orbital coupling scheme will be presented to rationalise this unique behaviour and also to account for the unusually long wavelenght, 1946 nm, of the haem charge-transfer (porphyrin → Fe III band. The optical properties of this metal pair, which lie at the active site of the enzyme, have been explored in a number of derivatives. The functional implications of these features are discussed by Dr. C. Greenwood in his paper. EPR-silent states of iron-sulphur proteins. The low temperature MCD spectra of iron-sulphur clusters in paramagnetic oxidation states are highly structures giving excellent means of identifying cluster type [2]. A number of examples will be given to illustrate the identification of cluster type and oxidation state in complex proteins. The MCD characterisation of proteins containing [3Fe-xS] centres will be described and the transformation of 3Fe to 4Fe clusters and vice versa will be shown [5]. A discussion of cluster type and properties in the proteins nitrogenase and hydrogenase will also be give [6].

The low temperature magnetic circular dichroism spectra of iron-sulphur proteins II. Two-iron ferredoxins

Variable temperature magnetic circular dichroism (MCD) spectra of a number of two-iron ferredoxins have been measured. The spectra of fully oxidised spinach and Spirulina maxima ferredoxin are independent of temperature between room temperature and 18 K, showing that no contribution to the room temperature MCD spectrum arises from the small population of low-lying excited states originating from the exchange coupling. However, the low temperature MCD spectra of the half-reduced proteins spinach and Spirulina maxima ferredoxin and adrenodoxin are all reasonably intense and temperature dependent. An interpretation of the spectrum of the charge-transfer region is suggested by starting with the assignments previously obtained from rubredoxin.

The low temperature magnetic circular dichroism spectra of iron-sulphur proteins I. Oxidised rubredoxin

Variable temperature magnetic circular dichroism spectra have been measured on oxidised Clostridium pasteurianum rubredoxin. Evidence has been obtained for the presence of two one-electron charge-transfer transitions, sulphur to ferric ion, in the region 15 000 to 28 000 cm −1. The first moment of the lower energy band is consistent with it being the orbital transition t 1 non-bonding sulphur orbital, to the 2 e ferric d-orbital. The magnitude of the spin-orbit coupling constant in the lower excited state has been determined and shown to be small compared with the axial distortion. The splitting of the low energy band observed in the absorption spectrum can therefor be equated directly with the axial distortion of the lowest excited charge-transfer state. Finally, the potential utility of making saturation experiments at very low temperatures has been examined.

Low temperature magnetic circular dichroism spectra of met- and myoglobin derivatives

1. 1.|Low temperature magnetic circular dichroism spectra of high and low spin derivatives of metmyoglobin and myoglobin have been measured in the Soret and high wavelength regions. 2. 2.|The large difference in intensity of the Soret magnetic circular dichroism bands suggest that a correlation exists between the signal intensity and spin state of the heme-iron. 3. 3.|From a comparison of the high and low spin spectra of the myoglobin derivatives it is concluded that oxymyoglobin contains between 10 and 20% of a ferrous high spin component below 100°K.

Magnetic circular dichroism assignment of the longest wavelength band of a series of tetrahedral d 0 OXY- and Thioanions

Room temperature magnetic circular dichroism spectra have been obtained for MoS 2− 4, MoO 2− 4, WS 2− 4, ReS − 4, VS 3− 4, VO 3− 4 and OsO 4 through their first and second charge-transfer bands. These measurements demonstrate that the band at longest wavelength (ν 1 band) must be assigned to a t 1 → 2e transition for all the compounds investigated.