©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Spectroelectrochemical Characterization of the Metal Centers in Carbon Monoxide Dehydrogenase (CODH) and Nickel-deficient CODH from Rhodospirillum rubrum(*)

(Received for publication, November 13, 1995; and in revised form, January 19, 1996)

Nathan J. Spangler (1) Paul A. Lindahl (2) Vahe Bandarian (1) Paul W. Ludden (1)(§)

From the  (1)Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706 and the (2)Department of Chemistry, Texas A & M University, College Station, Texas 77843

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum rubrum contains two metal centers: a Ni-X-[Fe(4)S(4)] cluster (C-center) that serves as the COoxidation site and a standard [Fe(4)S(4)] cluster (B-center) that mediates electron flow from the C-center to external electron acceptors. Four states of the C-center were previously identified in electron paramagnetic resonance (EPR) and Mössbauer studies. In this report, EPR-redox titrations demonstrate that the fully oxidized, diamagnetic form of the C-center (C) undergoes a one-electron reduction to the C state (g = 1.87) with a midpoint potential of -110 mV. The reduction of C to C is shown to coincide with the reduction of an [Fe(4)S(4)] cluster in redox-titration experiments monitored by UV-visible spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet contains both [Fe(4)S(4)] clusters, does not exhibit EPR-active states or reduced Fe(4)S(4) clusters at potentials more positive than -350 mV.


INTRODUCTION

Carbon-monoxide dehydrogenase (CODH) (^1)from the purple, nonsulfur bacterium Rhodospirillum rubrum is an oxygen-labile, nickel-containing enzyme that reversibly catalyzes the oxidation of CO to CO(2)(1) . Unlike CODHs from acetogenic and methanogenic bacteria, R. rubrum CODH is unable to catalyze the synthesis or degradation of acetyl-CoA(2, 3, 4, 5, 6, 15) . As purified, CODH is a 62-kDa monomer with 1 nickel atom and 8 iron atoms/ monomer(7) . A nickel-deficient form of CODH, which contains all of the iron components of holo-CODH yet has no CO-oxidation activity, is obtained by growing R. rubrum cultures on nickel-depleted medium(8, 9) . Treating nickel-deficient CODH with NiCl(2) produces an activated form of the enzyme that contains 1 mol of nickel/mol of enzyme and exhibits the same level of CO-oxidation activity as CODH purified from cells grown on nickel-supplemented medium (holo-CODH)(9) .

Holo-CODH contains only two metal centers, which have been designated as the B-center and C-center. Similar metal centers are also found in CODHs from methanogenic and acetogenic bacteria, including the well studied CODH from Clostridium thermoaceticum(10, 11, 12, 13, 14, 15) . The C-center, which serves as the CO-oxidation site(16, 17, 18, 19, 20) , is composed of an [Fe(4)S(4)] cluster (22) that is linked to nickel by an unidentified bridging ligand, X (Ni-X-[Fe(4)S(4)]) (19, 20, 21) . The B-center consists solely of a standard [Fe(4)S(4)] cluster (22) which mediates electron flow between the C-center and external electron acceptors(8, 17, 18) . Nickel-deficient CODH, which can only be obtained in R. rubrum, has the B-center and a [Fe(4)S(4)] cluster (referred to as the C*-center) that is a precursor to the C-center of holo-CODH. The Fe(4)S(4) clusters of nickel-deficient CODH cannot be reduced by CO(20) .

Four states of the C-center of holo-CODH have been characterized (Table 1, Fig. 1) ( (8) and references within). C and C have electronic spins S = and S = , respectively, and result from the reduction of holo-CODH with CO or dithionite. C and C are replaced by a second S = form, C, and a diamagnetic form, C, by the oxidation of CO- or dithionite-reduced holo-CODH with the redox dye indigo carmine (E`(o) = -125 mV). Oxidation with thionin (E`(o) = 56 mV) fully converts the C-center to the C form which is EPR silent. If all states of the C-center contain Ni(II)(20) , then C, C and C appear to be one-electron reduced forms of C(22) . C, however, has been proposed to be three electrons more reduced than C, therefore C may contain a more reduced form of nickel(11) .




Figure 1: Model for the redox states of the B- and C-centers of R. rubrum CODH. Approximate midpoint potentials of the redox couples are centered between their vertical positions in the figure. The C/C couple undergoes a reversible one-electron redox reaction with a midpoint potential of -110 mV. C/C appear to be separated by one electron with a midpoint potential below -400 mV. C may be one or three electrons more reduced than C. All forms of the C-center are thought to contain Ni(II), with the exception of C which may contain a more reduced form of nickel in the event that C is three electrons more reduced than C.



The C*-center of nickel-deficient CODH is stable in two forms (Table 1, Fig. 1)(22) . The C* state, with electronic spin S = , is a one-electron reduced form of the C*-center observed following reduction of nickel-deficient CODH with dithionite. The EPR signal originating from C* has broad resonances in the g = 4-6 region. This signal is very similar to the C EPR signal of holo-CODH. Following oxidation of reduced, nickel-deficient CODH with indigo carmine or thionin, the C* state is completely converted by a one-electron oxidation to the diamagnetic C* form.

The properties of the B-center in holo-CODH and nickel-deficient CODH are very similar(22) . The B-center is stable in only two forms (Table 1, Fig. 1)(22) . The B state develops following the reduction of nickel-deficient CODH with dithionite or the reduction of holo-CODH with CO or dithionite. By oxidizing either form of reduced CODH with indigo carmine or thionin, B is completely converted to B. The reversible oxidation of B to B in holo-CODH was previously shown to be a one-electron process with a midpoint potential of -418 mV(23) .

A significant problem in studying the nickel-containing CODHs has been that the spin concentrations of the C (g = 1.87) and C (g = 1.86) EPR-signals vary from one sample preparation to another (e.g. 0.05-0.5 mol of spin/mol of C-center), and are much lower than expected for isolated S = systems. Hu et al.(22) concluded that this observation derives from heterogeneity in the C-center population resulting from a mixture of spin and oxidation states; EPR and Mössbauer studies demonstrate a combination of C and C states in CO- or dithionite- reduced holo-CODH and a combination of C and C states in indigo carmine-oxidized holo-CODH. Interestingly, the ability to observe the C or C EPR signal does not correlate with enzyme activity; all preparations of purified CODH have similar specific activities. The factors affecting the states of the C-center are incompletely understood; therefore, this report investigates the effect of redox potential on the states of the C-center.

In this report, we have combined potentiometric redox titrations with UV-visible and EPR spectroscopies to study the redox characteristics of the metal centers in holo- and nickel-deficient CODH. The midpoint potential of the C/C redox couple has been determined and correlated with the reduction of an [Fe(4)S(4)] cluster in holo-CODH. Finally, this work demonstrates nickel-deficient CODH, which does not exhibit a C state, does not contain an [Fe(4)S(4)] cluster that can be reduced over the range of potentials that afford the C state in holo-CODH.


EXPERIMENTAL PROCEDURES

Cell Growth and Enzyme Purification

Cell cultures of R. rubrum were grown on medium that afforded either holo- or nickel-deficient CODH as needed, and enzyme purification was performed according to published methods(1, 8, 20) . Protein concentrations were determined by the bicinchoninic acid colorometric method using bovine serum albumin (grade A, Sigma) as standard(25) . CO-oxidation activity was determined by the CO-dependent methyl viologen reduction assay as described previously(20) . One unit of activity equals 1 µm CO-oxidized per minute.

EPR Spectroelectrochemistry

The redox potentials of CODH samples were poised in an anaerobic electrochemical cell (26) with a three-electrode system consisting of a silver/silver chloride reference electrode, a platinum counter electrode, and a gold working electrode. The following mediators were used in electrochemical experiments: methyl viologen (E`(o) = -440 mV), benzyl viologen (E`(o) = -358 mV), indigo carmine (E`(o) = -125 mV), indigo tetrasulfonate (E`(o) = -46 mV), and thionin (E`(o) = 56 mV)(13) . Methyl and benzyl viologens were purchased from Sigma; indigo carmine, indigo tetrasulfonate, and thionin were purchased from Aldrich. All mediators were used without further purification.

The electrochemical cell and redox solutions were prepared in an anaerobic glove box (Vacuum/Atmospheres Dri-Lab glovebox model HE-493) with an N(2) atmosphere containing less than 1 ppm O(2). The buffer used in all experiments was 100 mM MOPS, pH 7.2. Buffer solutions and the electrochemical cell were stored in the glove box prior to use. In a typical experiment, approximately 15-30 mg of CODH (with a specific activity of 4,200 units/mg) in a buffer solution containing 400 mM NaCl and 1 mM dithionite was chromatographed through a Sephadex G-25 gel-filtration column to remove dithionite. The dithionite-free CODH eluent was diluted to 3 ml with anaerobic buffer containing mediators (approximately 0.04 mM final concentration each) and KCl (0.1 M final concentration). This solution was added to the electrochemical cell, which was assembled and removed from the glove box in order to perform the potentiometric titrations. Once outside the glovebox, a continuous flow of scrubbed argon was passed through the electrochemical cell to maintain oxygen-free conditions. The redox potential was established with an Electrosynthesis model 410 Potentiostatic Controller, and once equilibrated (i.e. drift < 2 mV/min), the redox-poised solution was anaerobically transferred into an EPR tube, and frozen in liquid N(2). Fully reduced samples of holo-CODH and nickel-deficient CODH were prepared by exposure to CO, or by adding an excess of dithionite. EPR spectra were recorded on a Bruker ESP 300 or a Varian E-15 spectrometer using an Oxford Instruments ER910A cryostat. Relative signal intensities were fitted to a linearized form of the Nernst equation (), and the midpoint potential (E(o)) and the number of electrons (n) involved in a redox reaction were determined according to published methods(13) .

The data were plotted as signal intensity versus the measured potential (E(h)), and a theoretical curve was generated with n and E(h) values determined by Nernst analysis. Redox potentials are reported in reference to the normal hydrogen electrode.

UV-visible Spectroelectrochemistry

CODH samples were electrochemically titrated, as described above, in an electrochemical cell fused to a 1-cm path length quartz UV-visible cell. CODH activities were: 3,700 units/mg for holo-CODH; 23 units/mg for nickel-deficient CODH. UV-visible spectra were recorded on a Shimadzu UV-1601PC spectrophotometer. Mediator contributions to the combined CODH-mediator spectrum were accounted for according to published procedures(27) . Potentials more negative than -350 mV were stabilized by adding CO (5%) to the headspace of the redox vessel followed by flushing with argon. Reduction of Fe-S clusters in CODH was measured by the change in absorption at 420 nm. Measured A values were converted to percent oxidation by and plotted versus measured redox potential (28) .

CO- or dithionite-reduced CODH exhibited a minimum absorbance at 420 nm (A (minimum), = 20.1 mM cm), and thionin-oxidized CODH exhibited a maximum absorbance at 420 nm (A (maximum), = 35.6 mM cm)(8) .

Combined UV-vis/EPR-redox Titration

CODH samples were prepared in the anaerobic glove box. Dithionite was removed by G-25 chromatography, and dithionite-free CODH was diluted with anaerobic buffer (100 mM MOPS, pH 7.2) to a final concentration of 0.043 mM and divided into 0.2-ml aliquots. CODH activity was 4,000 units/mg. Several aliquots were added directly to EPR tubes (as-isolated CODH), and the remainder were treated with small amounts of either dithionite or thionin before transferring into EPR tubes. Fully oxidized CODH was prepared by adding a slight excess of thionin, which was detected by the presence of a light blue color. Fully reduced CODH was prepared (outside the glovebox) by flushing a sealed EPR tube containing as-isolated CODH with CO. After sealing and removing the EPR tubes containing CODH samples from the glove box, the visible spectra of these samples were recorded with a Shimadzu UV-1601PC spectrophotometer fitted with an adapter for holding an EPR tube in the sample cell. Immediately after recording an optical spectrum, the EPR tube was removed and immersed in liquid nitrogen to freeze the CODH sample for subsequent EPR spectroscopy. Percent oxidation of [Fe(4)S(4)] clusters was determined, and EPR spectroscopy was performed as described above.


RESULTS AND DISCUSSION

EPR-Redox Titrations of the Metal Centers in Holo- and Nickel-deficient CODH

EPR-redox titration experiments were performed to determine the optimum redox potential for observation of the C (g = 1.87) EPR signal. Additionally, the redox dependence of EPR-active states of the C*-center in nickel-deficient CODH as well as those of the B-center in holo-CODH and nickel-deficient CODH were investigated. At a potential of -33 mV, no significant EPR signals were observed for holo-CODH (Fig. 2, A). The C (g = 1.87) signal developed as potentials decreased (Fig. 2, B), and Nernst analysis indicated that the C-center undergoes a reversible, one-electron redox reaction with a midpoint potential of -110 mV (Fig. 2, lower panel). At potentials below -350 mV, the B (g = 1.94) state was observed with the loss of C (Fig. 2, C).


Figure 2: Upper panel, representative EPR spectra of holo-CODH from R. rubrum at different redox potentials. Purified CODH (6 mg/ml) was in 100 mM MOPS, pH 7.2, and 0.1 mM KCl. Redox potentials of samples were set as described under ``Experimental Procedures.'' Spectrometer conditions were: microwave power, 5 mW; modulation amplitude, 5 G; frequency, 9.236 GHz; temperature, 10 K. Spectra shown were recorded for samples poised at -33 mV, spectrum A; -315 mV, spectrum B; and -425 mV, spectrum C. Lower panel, EPR spectroelectrochemical titration of the C-center. The amplitude of the g = 2.03 resonance was followed as the redox potential was varied. Analysis by the Nernst equation yielded an E(o) of -110 mV and a slope of -62 mV.



The EPR-redox titration data presented in Fig. 2indicate that C (g = 1.87) results from a one-electron reduction of the C with a midpoint potential of -110 mV. The maximum spin concentration measured in this experiment for the g = 1.87 EPR signal is 0.2 mol of spin/mol of CODH; therefore, the formation of the C state appears to be influenced by factors other than redox potential alone. This observation is consistent with the findings of Hu et al., who report a mixture of C and C states in Mössbauer spectra of holo-CODH poised at potentials as low as -300 mV. Hu et al. concluded that, due to sample heterogeneity, a fraction of holo-CODH molecules may contain C-centers that are unable to form C. This fraction, therefore, is proposed to remain in the C state until potentials low enough to result in the formation of C(S) and C are achieved.

Nickel-deficient CODH did not exhibit EPR-active states in the g = 2 region (Fig. 3A) or g = 4-6 region (data not shown) at potentials more positive than -350 mV. Initial development of the B signal (g = 1.94) was observed at -400 mV (Fig. 3B). At -510 mV, however, nickel-deficient CODH exhibited a two-component spectrum consisting of B and a similar and overlapping signal with g values at 2.07, 1.93, and 1.86 (Fig. 3C). The nature of the second signal is unknown and is currently under investigation.


Figure 3: Representative EPR spectra of nickel-deficient CODH from R. rubrum at different redox potentials. Purified nickel-deficient CODH (10 mg/ml) was in 100 mM MOPS, pH 7.2, and 0.1 mM KCl. Redox potentials of samples were set as described under ``Experimental Procedures.'' Spectrometer conditions were as described in Fig. 1, except that the microwave power was 20 mW and the frequency was 9.460 GHz. Spectra shown were recorded for samples poised at -100 mV (spectrum A), -400mV (spectrum B), and -510 mV (spectrum C).



The C*-center does not appear to be redox active over the range of potentials that afford the C state. This observation is consistent with the proposal that an interaction between the nickel and [Fe(4)S(4)] components in the C-center strongly influences the redox behavior of the Fe(4)S(4) cluster. In the absence of nickel, therefore, reduction of the [Fe(4)S(4)] cluster of C* can only occur at potentials significantly lower than the E(o) = -110 mV of the C/C redox transition.

The B-centers in holo- and nickel-deficient CODH exhibited similar redox behavior. The reduced form of the B-center, B, is not observed at potentials more positive than -350 mV in EPR-redox titrations of either form of CODH ( Fig. 2and Fig. 3). The redox properties of the B-center, therefore, appear to be unaffected by nickel in holo-CODH. The absence of B at potentials above -350 mV is also consistent with the finding of Smith et al.(23) that the B-center in holo-CODH undergoes reduction with a midpoint potential of -418 mV.

Combined UV-visible and EPR Study of the Metal Centers in Holo-CODH

UV-visible spectroscopy was utilized in the following experiments to observe changes in the redox state of [Fe(4)S(4)] clusters in CODH by monitoring changes in absorbance at 420 nm (a wavelength sensitive to the redox state of Fe(4)S(4) clusters). The Fe(4)S(4) component of C was proposed to be in the one-electron reduced [Fe(4)S(4)] form(22) , and the C (g = 1.87) state was shown in this work to result from a one-electron reduction (E(o) = -110 mV) of C (Fig. 2). To demonstrate that reduction of [Fe(4)S(4)] clusters accompanies the development of the C EPR signal (g = 1.87), combined EPR and UV-visible spectroscopy experiments were performed.

The corresponding EPR and UV-visible spectra of fully oxidized holo-CODH had no significant EPR signals (Fig. 4A, upper panel) and a maximum absorbance at 420 nm (Fig. 4A, lower panel). This shows that all C-centers were in the C form and all [Fe(4)S(4)] clusters were oxidized. Reduction of 33% of the Fe(4)S(4) clusters in holo-CODH, as determined by A measurement (Fig. 4B, lower panel), was accompanied by the appearance of the C state (g = 1.87) in the corresponding EPR spectrum (Fig. 4B, upper panel). (The [Fe(4)S(4)] clusters of the B- and C-centers are assumed to contribute equally to the optical spectrum at 420 nm). As the percentage of reduced Fe(4)S(4) clusters increased beyond 33%, the EPR signal intensity of C (g = 1.87) did not increase. With 56% of the Fe(4)S(4) clusters reduced in holo-CODH, partial development of the B state (g = 1.94) was evident (Fig. 4C, upper and lower panels). Fully reduced holo-CODH exhibited EPR signals originating from the B and C (g = 1.86) states (Fig. 4D, upper panel) and a minimum absorbance at 420 nm (Fig. 4D, lower panel).


Figure 4: EPR and UV-visible spectra of holo-CODH samples at various levels of Fe-S cluster reduction determined by A. A dithionite-free sample of ``as-isolated'' CODH (3 mg/ml) in 100 mM MOPS, pH 7.2, was obtained as described under ``Experimental Procedures.'' The C state and 100% oxidation of Fe-S clusters were obtained by adding a slight excess of thionin (traces A); the C state (g = 2.03, 1.88 and 1.71) and 33% reduction of Fe-S clusters in ``as-isolated'' CODH were obtained without addition of oxidant or reductant (traces B); the C and B (g = 2.04, 1.97 and 1.88) states and 66% reduction of Fe-S clusters were obtained following the addition of a small amount of sodium dithionite (traces C); the B and C (g = 1.97, 1.88 and 1.75) states and full reduction of Fe-S clusters were obtained after incubation under CO (traces D). EPR conditions were as described in Fig. 1.



The data in Fig. 4correlates the development of the C EPR signal (g = 1.87) with a decrease in A, which is consistent with the proposal that C results from the one-electron reduction of the [Fe(4)S(4)] component of C. The development of the lower-potential B state was accompanied by additional loss of absorbance at 420 nm, and C was not observed until all [Fe(4)S(4)] clusters became reduced. Although present in holo-CODH, nickel does not contribute to the optical spectrum at 420 nm(8) .

Optical-Redox Titration of the Metal Centers in Holo- and Nickel-deficient CODH

The midpoint potentials for the reduction of [Fe(4)S(4)] clusters in holo-CODH and nickel-deficient CODH were determined by optical-redox titration experiments. The results (Fig. 5) show that 30% of the Fe(4)S(4) clusters in holo-CODH (i.e. 60% of the C-centers) undergoes a one-electron reduction with a midpoint potential of approximately -125 mV, similar to that measured for the C/C redox transition. This is consistent with the observation of Hu et al. that only a fraction of the C-center population (no greater than 60% under any known condition) is capable of forming C(22) .


Figure 5: UV-visible spectroelectrochemical titration of the Fe-clusters in CODH. The intensity of the absorbance at 420 nm was followed as the redox potential was varied. Solid circles (holo-CODH) and open circles (nickel-deficient CODH) represent the fractional absorbance changes at 420 nm expressed in percentages. The theoretical lines drawn through the points are Nernst n = 1 curves, E(o) = -415 mV for nickel-deficient CODH; E(o) = -415 mV, and E(o) = -125 mV for holo-CODH.



Beginning at approximately -350 mV, decreases in A were observed for holo-CODH and nickel-deficient CODH, and the minimum A absorption value for each sample was achieved at -500 mV, indicating full reduction of all [Fe(4)S(4)] clusters (Fig. 5). Notably, significant reduction of Fe(4)S(4) clusters in nickel-deficient CODH was not observed at potentials greater than -350 mV. This observation, coupled with the absence of the C* EPR signal at similar potentials, shows that the fully oxidized form of the C*-center (C*) is not redox active at potentials greater than -350 mV.

Although this work focuses primarily on the redox properties of [Fe(4)S(4)] clusters that in part define the various states of the B- and C-centers, the role of nickel in the C-center should not be overlooked. The presence of nickel dramatically increases the midpoint potential of the C-center [Fe(4)S(4)] cluster by over 200 mV. Moreover, Hu et al.(22) found evidence in Mössbauer studies that the [Fe(4)S(4)] component of the C state contains a unique pentacoordinate iron subsite, called ferrous component II(29) , which was not observed in the C*-center; the spectroscopic properties of the C*-center Fe(4)S(4) cluster were consistent with all iron atoms having tetracoordination(22) . Treating nickel-deficient CODH with nickel converts the C*-center to the C-center coincident with the development of the unique ferrous component II subsite; therefore, the incorporation of nickel appears to substantially alter the coordination environment of one iron atom in the C-center. The reduced [Fe(4)S(4)] cluster in the C state, therefore, may be stabilized by electrostatic interactions from the nickel cation or from an alteration of the ligands and protein environment of the C-center Fe(4)S(4) cluster when nickel is bound to CODH.

It is interesting to note that the activation of nickel-deficient CODH by NiCl(2) occurs only at potentials lower than -350 mV(9) . From the results presented here, this suggests that the C*-center must be in the C* state (and perhaps the B-center in the B state) before the appropriate ligand environment is accessible to the nickel cation.

The nickel ion appears to remain as Ni(II) in the various states of the C-center studied here. Evidence of a Ni(III) EPR signal was not observed at any potential. Furthermore, the correlation established between the optical changes monitored at 420 nm and the development of the C (g = 1.87) EPR signal demonstrates that the one-electron reduction of C that affords C is localized on the [Fe(4)S(4)] component of the C-center. The redox behavior of the C-center at potentials more negative than -350 mV is unclear, as is the relationship between CO-oxidation activity and the appearance of the C, C and C(S) states. Both the C and C forms of the C-center must be competent to bind and be reduced by CO as holo-CODH containing either form of the C-center can be fully reduced by CO. It is possible that multiple ``routes'' of reduction exist among the four states of the C-center, but data presented here do not allow us to distinguish such possibilities. Finally, the number of electrons (i.e. one or three) separating C from C is unknown.

In summary, the C/C redox couple has a midpoint potential of -110 mV. The formation of C from C is concomitant with the one-electron reduction of a [Fe(4)S(4)] cluster in holo-CODH. The nickel cation, proposed to be Ni(II) in the C and C states, appears to strongly affect the redox behavior of the [Fe(4)S(4)] component of the C-center; reduction of the C*-center [Fe(4)S(4)] cluster in nickel-deficient CODH is not observed at potentials more positive than -350 mV.


FOOTNOTES

*
This work was supported by Grant DE-FG02-F7 EX 13691 from the United States Department of Energy (to P. W. L.) and Grant GM46441 from the National Institutes of Health (to P. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 608-262-6859; Fax: 608-262-3453; ludden{at}biochem.wisc.edu.

(^1)
The abbreviations used are: CODH, carbon-monoxide dehydrogenase; MOPS, 4-morpholinepropanesulfonic acid; W, watt(s).


ACKNOWLEDGEMENTS

We thank Eckard Münck and Zhengguo Hu as well as Brian Fox for helpful discussions regarding spectroscopic characterizations and redox-titration techniques. We also thank George Reed for the generous use of his EPR spectrometer.


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