Metal Clusters in Proteins - American Chemical Society

Chapter 16

Variable-Temperature Magnetic Circular Dichroism Studies of Metalloproteins Michael K. Johnson

Downloaded by FUDAN UNIV on February 15, 2017 | http://pubs.acs.org Publication Date: June 21, 1988 | doi: 10.1021/bk-1988-0372.ch016

Department of Chemistry, University of Georgia, Athens, GA 30602 Variable temperature magnetic circular dichroism spectroscopy affords a means of investigating the electronic and magnetic properties of metal centers in metalloproteins. This pedagogical account discusses the experimental and theoretical aspects of the technique that are applicable to the study of metal centers and metal clusters in proteins. The type of information that is available includes resolving and assigning electronic transitions and determining ground state parameters such as spin state, g-values, zero field splitting parameters. These aspects are illustrated using Ni(II)-substituted rubredoxin, the [3Fe-4S] cluster in a bacterial ferredoxin, and the MoFe and VFe nitrogenase proteins as examples. Magnetic circular dichroism (MCD) spectroscopy is being increasingly used to investigate the electronic and magnetic properties of metal centers in metalloproteins (for recent reviews see Refs. 1-3). The technique facilitates resolution and assignment of electronic transitions and is applicable to any chromophoric metal center. Moreover, since paramagnetic metal centers invariably afford temperature-dependent MCD bands, variable temperature studies enable selective identification and deconvolution of the optical transitions from individual chromophores, even in complex multicomponent metalloenzymes such as cytochrome c oxidase (4,5), succinate dehydrogenase (6), and nitrogenase (7,8). When paramagnetic metal centers are present, careful analysis of the temperature and/or magnetic field dependence of discrete transitions furnishes electronic ground state information such as spin state, g-values, zero field splitting parameters, and magnetic coupling constants. While similar information is often accessible via EPR, Mdssbauer, or magnetic susceptibility studies, MCD offers certain advantages over each of these techniques in the study of metalloproteins. For example, unlike Mflssbauer, it is not limited to Fe and does not require isotopic enrichment. Weak magnetic interactions or zero field splittings that can prevent the observation of EPR resonances, do not prevent characterization of paramagnetic center(s) by MCD spectroscopy. Magnetic susceptibility studies can be difficult to interpret for multicentered metalloproteins particularly when paramagnetic impurities are present, whereas with MCD spectroscopy the magnetic properties of individual centers can be investigated independently. However, it should be emphasized that MCD studies

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Que; Metal Clusters in Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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are usually most informative when used in combination with one or more of the above techniques. This pedagogical account is intended to provide a brief introduction for the non-specialist, to the theoretical and experimental aspects of variable temperature MCD spectroscopy that are applicable in the study of metalloproteins. This is followed by some individual examples of MCD studies of metalloproteins that have been chosen to illustrate the utility of the technique and the type of information that is available.


Theoretical Aspects Electronic transitions between Zeeman components of the electronic ground and excited states are circularly polarized. MCD exploits this phenomena, measuring the differential absorption of left and right circularly polarized light, A A, as function of wavelength in the presence of a magnetic field applied parallel to the direction of light propagation. This difference technique, as opposed to the conventional Zeeman experiment, is necessary because the absorption band widths observed for metalloproteins are far too large to permit resolution of Zeeman splittings. Theoretical treatments (see for example ref. 9) lead to a general expression for MCD intensity for a transition A —• J: (1)

where 7 is a collection of physical constants, b is the molar concentration, 1 is the pathlength (cm), B is the magnetic flux, p is the Bohr magneton, f(E ,E) is the line shape which is a function of both the transition energy, JA = J - A» * the incident photon energy, E, and A B , and C are parameters that depend on the electric dipole selection rules for the absorption of circularly polarized light in a magnetic field. From this expression, it is apparent that MCD intensity is the sum of three terms, -A^df/dE), B f, and C f/kT, which are called A, B, and C-terms, respectively. In most cases, A, B, and C-terms may be distinguished by their dispersion and temperature dependence. A-terms arise when there is degeneracy in either the excited state only, or both the ground and excited states. They are temperature independent and give rise to derivative-shaped dispersion with a cross-over point at the energy of the zero field absorption maximum. B-terms originate from field-induced mixing of the ground and excited state with all other possible excited states. While non-overlapping B-terms will exhibit absorption-shaped dispersion, derivative-shaped B-terms (pseudo-A-terms) can result from overlapping transitions with oppositely signed B parameters. B-terms will be temperature independent, except in the rare occurrence of a mixing state that becomes thermally populated over the temperature range of the experiment. C-terms require a degenerate or near-degenerate ground state. In common with B-terms, non-overlapping C-terms will exhibit absorption-shaped dispersion. However, when the excited state is split by an energy smaller than the bandwidth and the C-terms to each component are of opposite sign, derivative-shaped bands (pseudo-A-terms) can appear. This situation is frequently encountered for excited states that are split by spin-orbit coupling. Fortunately C-terms are readily distinguished from A- and B-terms by their inverse temperature dependence. If present, they usually dominate the MCD spectrum at low temperatures, since they can be enhanced up to 70-fold on going from room temperature to liquid helium temperatures. For biological chromophores, which generally possess only low symmetry, ground state degeneracy can JA

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usually be equated with spin degeneracy. Consequently low temperature MCD provides a selective optical probe for paramagnetic chromophores such as transition metal centers. Equation 1 only applies to paramagnetic chromophores in the Curie law-limit, i.e. kT much greater than the ground state Zeeman splitting, g0B. However, on lowering the temperature or increasing the field so that gfiB becomes comparable or greater than kT, AA will become non-linear as a function of B/T and eventually independent of B/T. At this point only the lowest Zeeman component is populated and the system is completely magnetized. Plots of AA as a function of B/T to magnetic saturation are termed MCD magnetization plots. Analysis of such plots, after correction for temperature independent contributions, can lead to estimates of ground state g-values and hence spin state. The theory for analyzing MCD magnetization plots is well developed for ground states consisting of an isolated' Kramers doublet (i.e. no low lying states becoming thermally populated over the temperature range of the experiment) (10-12). While a detailed discussion of the theoretical expressions used in fitting magnetization data is beyond the scope of this article, it is important to realize that, unlike magnetization curves derived from magnetic susceptibility data, the steepness of MCD magnetization plots is dependent on the polarization of the electronic transition as well as the ground state g-values. Indeed the dependence on the polarization becomes particularly acute for ground states with large g-value anisotropy (12,13). So far the discussion has been confined to 'isolated' Kramers doublet ground states and therefore is strictly applicable only to non-interacting centers with S - 1/2 ground states. Paramagnetic transition metal centers with S > 1/2 ground states are readily recognizable by their MCD magnetization characteristics. First, the curves deviate substantially from theoretical data for g - 2. Second, data points measured at different temperatures do not lie on a smooth curve, due to the population of low lying zero field components. Third, plots of MCD intensity as a function of 1/T only become linear at high temperatures when the spread of zero field components is very much less than kT. Complete analysis of magnetization data from S > 1/2 chromophores presents a complex theoretical problem, requiring the inclusion of field induced mixing of the zero field components, as well as zero field splitting parameters, g-values, and the polarization of the transitions from each doublet. Preliminary analysis can be effected by fitting the data at a temperature such that only the lowest doublet state is populated, using the expressions derived for an 'isolated' doublet ground state. While such a procedure neglects field induced mixing of zero field components, it does permit estimates of the effective g-values for the lowest doublet and hence the ground state spin. Many transition metal centers exhibit extremely low lying energy states as a result of either zero field splitting or magnetic interaction. Provided the energies of these low lying states are in the range 1-100 cm" , they can be assessed by measuring the MCD intensity at small applied fields as a function of 1/T and fitting to a Boltzmann population distribution (14). In this way MCD studies afford quantitative assessment of zero field splitting parameters and/or magnetic coupling constants. av

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Experimental Aspects Instrumentation for MCD measurements consists of a spectropolarimeter mated to either an electromagnet or superconducting magnet. Commercial spectropolarimeters are available for the wavelength range 180 - 2000 nm and recent technological advances have enabled home-built instruments to go out to 5000 nm. To obtain electronic ground state information via magnetization curves, it is



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necessary to use magnetic fields of 5 Tesla or more, with sample temperatures as low as 1.5 K. Superconducting magnets that facilitate such experiments are available commercially from Oxford Instruments. With such high magnetic fields, it is necessary to separate the magnet spatially from the spectrometer and to shield certain components such as the photomultiplier tube and the photoelastic modulator. Accurate control and absolute measurement of both the temperature and magnetic field are essential and are accomplished by multiple resistance thermometers (such as carbon glass resistors which exhibit negligible magnetic field effects) mounted both above and below the sample, and by a transverse Hall probe positioned in place of the sample. The MCD experiment involves the detection of transmitted light, and consequently frozen samples must be in the form of a glass. With protein solutions this is achieved by addition of at least 40% (v/v) ethylene glycol, glycerol, or sucrose. It is essential to check that no spurious effects result from the medium, by comparing the spectroscopic properties (i.e. UV-visible absorption, room temperature MCD and CD, and EPR) and, wherever possible, the activity of the metalloprotein, both with and without the addition of the glassing agent. While in general the presence of the glassing agent does not appear to perturb the properties of the metal chromophore in metalloproteins, there are some notable exceptions. For example, the [4Fe-4S] center in nitrogenase Fe-protein consists of a mixture of species with S = 1/2 and 3/2 ground states in aqueous buffer solutions, whereas the addition of 50% ethylene glycol results in conversion of almost all of the clusters to the S = 1/2 form (15). It is not possible to generalize concerning the quantity of sample that is required for variable temperature MCD studies, since the intensity of the signals from different types of biological chromophores can vary by as much as three orders of magnitude. However, typically 200 - 300 ul of sample exhibiting a maximum OD in the wavelength region of interest of approximately 0.5, are usually required to produce optimal data. Sample cells for low temperature experiments consist of 0.1 cm or 0.2 cm quartz cuvettes that can be filled under anaerobic or aerobic conditions. The measured spectra are a superposition of the natural and magneticallyinduced CD. Unless otherwise indicated, published MCD spectra are shown after computer subtraction of the natural CD, with the intensity expressed as Ae in units of M" cm" , which corresponds to the difference in the molar extinction coefficients for left and right circularly polarized light. In the Curie law limit the Ac scale is often normalized per unit field, i.e. Ae/B. However, this procedure is not meaningful at high fields and low temperatures where saturation effects may be occurring, and in such cases the applied magnetic field is stated in the text or figure legend. Quantitation of the MCD intensity of frozen samples is accurate to ±10%. The major sources of error reside in changes in sample pathlength on freezing and depolarization of the light beam as a result of inhomogeneities in the frozen sample. The latter can be approximately assessed and corrected for measuring the CD of an optically active sample with and without the sample in position. 1+

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Examples The results of variable temperature MCD studies of Ni(II)-substituted rubredoxin, the 7Fe ferredoxin from Thermus thermophilus and the MoFe and VFe nitrogenase proteins from Azotobacter vinelandii are discussed below. These specific examples have been chosen to illustrate how the technique can be used to, (a) resolve and assign overlapping electronic transitions, (b) determine ground state g-values and spin state, (c) provide quantitative information concerning the energies of low lying states, and (d) deconvolute the optical transitions from individual chromophores in multicomponent metalloproteins.



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NidlVsubstituted rubredoxin. In view of the propensity of Ni(II) to form square planar complexes with chelating ligands possessing sulfhydryl groups, it was of interest to determine whether or not the constraints imposed by folding of the rubredoxin polypeptide are sufficient to afford tetrahedral coordination geometry. Since square planar and tetrahedral coordination geometry for Ni(II) give rise to diamagnetic (S = 0) and paramagnetic (S = 1) ground states, respectively, this question can readily be addressed by variable temperature MCD spectroscopy. Figure 1 shows the room temperature absorption spectrum and MCD spectra recorded at temperatures between 4.2 K and 137 K with a magnetic field of 4.5 T for Ni(II)-substituted rubredoxin from Desulfovibrio gigas in the wavelength region 300 - 800 nm. The room temperature absorption spectrum is composed of two intense S -> Ni(II) charge transfer bands centered at 360 nm and 450 nm and two weaker Ni(II) d-d bands at 670 nm and 720 nm. The corresponding MCD spectra exhibit bands in analogous regions that display unusual temperature dependence. Below 15 K, the MCD spectrum is independent of temperature, with all bands showing linear dependence on the magnetic field strength. Such behavior shows that a non-degenerate state lies lowest in energy. Based on their dispersion and intensity, the MCD bands originating from this non-degenerate level are assigned to B-terms. However, pronounced changes in the MCD spectrum occur at temperatures above 15 K. The negative bands at 437 nm and 718 nm and the positive bands at 338 nm and 363 nm all decrease with increasing temperature, concomitant with the appearance of a positive band at 437 nm. As shown in Figure 2, the negative band at 664 nm shows an initial increase and subsequent decrease with increasing temperature, with the maximum occurring around 50 K. This complex temperature dependence can readily be interpreted in terms of population of a degenerate low lying state that gives rise to C-terms exhibiting inverse temperature dependence. The energy separation between the non-degenerate and degenerate states can be assessed by fitting the MCD intensity as a function of 1/T at a discrete wavelength to a Boltzmann population distribution for a two level system. Figure 2 shows the temperature dependence of the MCD intensity at 664 nm, with the solid line corresponding to the best fit. The best fit data are for an energy separation of 55 cm" , and assume a two-fold degenerate excited state. Since only two variable parameters are involved (i.e. the energy separation and the magnitude of the C-term from the degenerate level) and the data at this wavelength display temperature dependence with a maximum around 50 K due to opposite signs for the C and B-terms, a unique fit is readily obtained. Similar values for the energy separation can be obtained by fitting 1/T dependence data at other wavelengths, where the C and B-terms from the two levels are of the same sign and have comparable intensity. Using this energy separation, the temperature-independent and temperature-dependent MCD spectra, originating from the non-degenerate and low-lying degenerate states, respectively, can readily be deconvoluted (data not shown). The presence of a low-lying degenerate state strongly suggests an S = 1 ground state. Accordingly, the coordination geometry is tetrahedral with a T ground state to a first approximation. Removal of the ground state degeneracy in zero field to yield a non-degenerate ground state could result from spin orbit coupling alone or a combination of spin orbit coupling and low symmetry distortions. While it is difficult to decide between these alternatives based on the MCD data for Ni(II)-rubredoxin alone, parallel studies of the analog complex [Ni(SPh) ] " (data not shown) strongly suggest the latter alternative. Except for the S Ni(II) charge transfer region being shifted to lower energy and a somewhat smaller energy separation (44 cm" ) between the non-degenerate 1

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Wavelength/nm Figure 1. Room temperature absorption and low temperature MCD spectra of Ni(II)-substituted rubredoxin from Desulfovibrio gigas. Arrows indicate the direction of change of MCD intensity with increasing temperature. Taken from Ref. 16.


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Figure 2. Temperature dependence of the MCD intensity of Ni(II)substituted rubredoxin from Desulfovibrio gigas at 664 nm. Taken from Ref. 16.


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and degenerate levels, the analog complex displays very similar MCD characteristics. Since X-ray crystallographic data for [Ni(SPh) ] ' show a D distorted NiS core (17), a similar distortion may be inferred for Ni(II)-substituted rubredoxin. The ground state is therefore split into A and E components, with the former lying lower in energy and exhibiting large, positive axial zero field splitting. In terms of the spin Hamiltonian the energy separation between the M = 0 and M = ±1 levels corresponds to the axial zero field splitting parameter D. The d-d region of the absorption spectrum is therefore assigned, under D symmetry to the A -+ A and A - • E components of the parent tetrahedral transition T (F) ^(P) transition. This example provides a good illustration of how variable temperature MCD measurements can be used to obtain zero field splitting parameters for nonKramers ground states with D > 0. For ground states where a doublet state lies lowest in energy (i.e. Kramers systems or axial non-Kramers systems with D < 0), the general procedure involves fitting plots of MCD intensity versus 1/T at small applied fields (i.e. in the Curie law limit) to a Boltzmann population distribution over the ground state manifold, with the axial zero field splitting and the C-terms from each doublet as the variable parameters (14). The accuracy of this procedure depends on the number of parameters and the extent of deviation from linearity of the plots of MCD intensity versus 1/T. 2

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7Fe ferredoxin from Thermits thermophilus. Iron-sulfur proteins provide good examples of the utility of variable temperature MCD spectroscopy for resolving electronic transitions and determining ground state properties (1). This is well illustrated by MCD studies of the oxidized and reduced [3Fe-4S] cluster in the 7Fe ferredoxin from T. thermophilus (13). Figures 3(a) and 3(b) show room temperature absorption spectra and variable temperature MCD spectra for the oxidized ([3Fe-4S] , [4Fe-4S] ) and partially-reduced ([3Fe-4S]°, [4Fe-4S] ) ferredoxin, respectively. The shoulder at 310 nm in the absorption spectrum of the partially reduced sample is due to excess of the reductant, dithionite. In both redox states the tetranuclear cluster has a S = 0 ground state, as a result of antiferromagnetic coupling, with the lowest paramagnetic level > 100 cm" higher in energy. Consequently, while both clusters contribute to the broad and featureless UV-visible absorption spectra, the temperature dependent C-terms that are observed can be attributed exclusively to the trinuclear center, which therefore must be paramagnetic in both the oxidized and one electron reduced states. The complexity of the low temperature MCD spectra of the oxidized and reduced trinuclear cluster shows the multiplicity of the predominantly S Fe charge transfer transitions that contribute to the absorption envelope. While MCD spectroscopy provides a method of resolving the electronic transitions, assignment cannot be attempted without detailed knowledge of the electronic structure. However, the complexity of the low temperature MCD spectra is useful in that it furnishes a discriminating method for determining the type and redox state of protein bound iron-sulfur clusters. Each well characterized type of iron-sulfur cluster, i.e. [2Fe-2S], [3Fe-4S], and [4Fe-4S], has been shown to have a characteristic low temperature MCD spectrum in each paramagnetic redox state (1) Inspection of the temperature dependence of the MCD bands of the oxidized and reduced [3Fe-4S] center in T. thermophilus ferredoxin, see Figure 3, reveals that the reduced cluster magnetizes more rapidly with decreasing temperature. This is shown more clearly in the magnetization data, which are shown as plots of MCD intensity, in the form of a percentage of that estimated 1+

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Metal Clusters in Proteins - American Chemical Society

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