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Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O–O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O–O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion o...

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O(2) carriers, we recently found that NO and O(2) binding in Thermus thermophilus (Tt) ba(3) is ~10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type Tt ba(3) and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O(2) channel of Tt ba(3) are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa(3) enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba(3) and the T231F mutant. The results show that the Tt ba(3) Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic "way station", which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba(3) and bovine aa(3) show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba(3) Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

We use a form of "freeze-trap, kinetic crystallography" to explore the migration of Xe atoms away from the dinuclear heme a(3)/Cu(B) center in Thermus thermophilus cytochrome ba(3) oxidase. This enzyme is a member of the heme-copper oxidase superfamily and is thus crucial for dioxygen-dependent life. The mechanisms involved in the migration of oxygen, water, electrons, and protons into and/or out of the specialized channels of the heme-copper oxidases are generally not well understood. Pressurization of crystals with Xe gas previously revealed a O(2) diffusion channel in cytochrome ba(3) oxidase that is continuous, Y-shaped, 18-20 Å in length and comprised of hydrophobic residues, connecting the protein surface within the bilayer to the a(3)-Cu(B) center in the active site. To understand movement of gas molecules within the O(2) channel, we performed crystallographic analysis of 19 Xe laden crystals freeze-trapped in liquid nitrogen at selected times between 0 and 480 s while undergoing outgassing at room temperature. Variation in Xe crystallographic occupancy at five discrete sites as a function of time leads to a kinetic model revealing relative degrees of mobility of Xe atoms within the channel. Xe egress occurs primarily through the channel formed by the Xe1 → Xe5 → Xe3 → Xe4 sites, suggesting that ingress of O(2) is likely to occur by the reverse of this process. The channel itself appears not to undergo significant structural changes during Xe migration, thereby indicating a passive role in this important physiological function.

Bacterial heme−copper terminal oxidases react quickly with NO to form a heme−nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N2O. Previously, we characterized the heme a3−NO complex formed in cytochrome ba3 from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to CuB(I) to form an end-on NO−CuB or a side-on copper−nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme−copper active site. Here we present a comparative study with cytochrome bo3 from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N2O. The EPR and resonance Raman signatures of the heme o3−NO complex are comparable to those of the a3−NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o3−NO complex does not produce a CuB−nitrosyl complex, but that instead, the NO remai...

In respiring organisms cytochrome c oxidase catalyzes proton translocation coupled to the reduction of O2 to water (1, 2). Cytochrome ba3 oxidase is one of two heme-copper oxidases isolated from T. thermophilus (3). It contains the dinuclear CuA center; a six-coordinated, low-spin, (6cLS) heme-B; and a (usually) five-coordinated, high-spin (5cHS) heme-As in close proximity to CuB ((3, 4) and references therein). The latter (heme-As and CuB) constitute the bimetallic site, and it is widely agreed that dioxygen binds to this site as the first step in O2-reduction and proton pumping. Two crystal structures of native and recombinant, oxidized ba3-type cytochrome c oxidase have been solved at

The structure of the soluble Rieske protein from Thermus thermophilus has been determined at a resolution of 1.3 A at pH 8.5 using multiwavelength anomalous dispersion (MAD) techniques. This is the first report of a Rieske protein from a menaquinone-utilizing organism. The structure shows an overall fold similar to previously reported Rieske proteins. A novel feature of this crystal form appears to be a shared hydrogen between the His-134 imidazole ring ligated to Fe2 of the [2Fe-2S] cluster and its symmetry partner, His-134', one being formally an imidazolate anion, Fe2-(His-134)N(epsilon)(-)...H-N(epsilon')(His-134')-Fe2', in which crystallographic C(2) axes pass equidistant between N(epsilon)...N(epsilon') and normal to the line defined by N(epsilon)...N(epsilon'). This provides evidence for a stable, oxidized cluster with a His(-) ligand and lends support to a previously proposed mechanism of coupled proton and electron transfer. A detailed comparison of the Thermus Rieske protein with six other Rieske and Rieske-type proteins indicates: (a) The cluster binding domain is tightly conserved. (b) The 3-D structure of the 10 beta-strand fold is conserved, even among the most divergent proteins. (c) There is an approximately linear relation between acid-pH redox potential and number of H-bonds to the cluster. (d) These proteins have two faces, one points into the larger complex (bc(1), b(6)f, or other), is involved in the proton coupled electron transfer function, and is highly conserved. The second is oriented toward the solvent and shows wide variation in charge, sequence, length, hydrophobicity, and secondary elements in the loops that connect the beta-sheets.

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA. Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.

Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.

The functioning of cytochrome

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdottir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.

Bacterial heme-copper terminal oxidases react quickly with NO to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second NO molecule to produce N(2)O. Previously, we characterized the heme a(3)-NO complex formed in cytochrome ba(3) from Thermus thermophilus and the product of its low-temperature illumination. We showed that the photolyzed NO group binds to Cu(B)(I) to form an end-on NO-Cu(B) or a side-on copper-nitrosyl complex, which is likely to represent the binding characteristics of the second NO molecule at the heme-copper active site. Here we present a comparative study with cytochrome bo(3) from Escherichia coli. Both terminal oxidases are shown to catalyze the same two-electron reduction of NO to N(2)O. The EPR and resonance Raman signatures of the heme o(3)-NO complex are comparable to those of the a(3)-NO complex. However, low-temperature FTIR experiments reveal that photolysis of the heme o(3)-NO complex does not produce a Cu(B)-nitrosyl complex, but that instead, the NO remains unbound in the active-site cavity. Additional FTIR photolysis experiments on the heme-nitrosyl complexes of these terminal oxidases, in the presence of CO, demonstrate that an [o(3)-NO.OC-Cu(B)] tertiary complex can form in bo(3) but not in ba(3). We assign these differences to a greater iron-copper distance in the reduced form of bo(3) compared to that of ba(3). Because this difference in metal-metal distance does not appear to affect the NO reductase activity, our results suggest that the coordination of the second NO to Cu(B) is not an essential step of the reaction mechanism.

Cytochrome ba3 is a cytochrome c oxidase from the plasma membrane of Thermus thermophilus and is the preferred terminal enzyme of cellular respiration at low dioxygen tensions. Using cytochrome ba 3 crystals pressurized at varying conditions under Xe or Kr gas, and X-ray data for six crystals, we identify the relative affinities of Xe and Kr atoms for as many as seven distinct binding sites. These sites track a continuous, Y-shaped channel, 18-20 A in length, lined by hydrophobic residues, which leads from the surface of the protein where two entrance holes, representing the top of the Y, connect the bilayer to the a3-CuB center at the base of the Y. Considering the increased affinity of O2 for hydrophobic environments, the hydrophobic nature of the channel, its orientation within the bilayer, its connection to the active site, its uniform diameter, its virtually complete occupation by Xe, and its isomorphous presence in the native enzyme, we infer that the channel is a diffusion pathway for O2 into the dinuclear center of cytochrome ba3. These observations provide a basis for analyzing similar channels in other oxidases of known structure, and these structures are discussed in terms of mechanisms of O2 transport in biological systems, details of CO binding to and egress from the dinuclear center, the bifurcation of the oxygen-in and water-out pathways, and the possible role of the oxygen channel in aerobic thermophily.

Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome c(552) in the cytoplasm of Escherichia coli yields both dimeric (rC(557)) and monomeric (rC(552)) cytochrome c-like proteins [Keightley, J. A., et al. (1998) J. Biol. Chem. 273, 12006-12016], which form spontaneously without the involvement of cytochrome c maturation factors. Cytochrome rC(557) is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276, 6537-6544]. Unexpectedly, the monomeric rC(552) transforms spontaneously to a cytochrome-like chromophore having, in its reduced state, the Q(oo) transition (alpha-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC(552), at 1.41 A resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH(2)-S-CH(2)-C(alpha)] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 A resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Q(oo)() transition, the presence of a new, high-frequency band in the resonance Raman spectra at 1666 cm(-1) for oxidized and at 1646 cm(-1) for reduced samples, and the greatly altered, paramagnetically shifted (1)H NMR spectrum observed for this species. The overall process defines a novel mechanism for oxidation of the 2-vinyl group to a 2-formyl group and adds to the surprising array of chemical reactions that occur in the interaction of heme with the CXXCH sequence motif in apocytochromes c.

The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide.

Recently, the genes of cytochrome ba_3 from Thermus thermophilus [Keightley, J. A., et al. (1995) J. Biol. Chem. 270, 20345−20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (Cu_A) domain of cytochrome ba_3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The Cu_A-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the Cu_A site in cytochrome ba_3 is a binuclear center. The absorption spectrum of the Cu_A site, free of the heme interference in cytochrome ba_3, is similar to the spectra of other soluble fragments from the aa_3-type oxidase of Paracoccus denitrificans [Lappalainen, P., et al. (1993) J. Biol. Chem. 268, 26416−26421] and the caa_3-type oxidase of Bacillus subtilis [von Wachenfeldt, C., et al. (1994) FEBS Lett. 340, 109−113]. There are intense bands at 480 nm (3100 M^(-1) cm^(-1)) and 530 nm (3200 M^(-1) cm^(-1)), a band in the near-IR centered at 790 nm (1900 M^(-1) cm^(-1)), and a weaker band at 363 nm (1300 M^(-1) cm^(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly β-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa_3 molecule [Iwata, S., et al. (1995) Nature 376, 660−669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 92, 11955−11959]. However, the thermostability of the fragment described here (T_m ≈ 80 °C) and the stable binding of copper over a broad pH range (pH 3−9) suggest this protein may be uniquely suitable for detailed physical-chemical study.

The local structure of the iron site in ferric superoxide dismutase from Escherichia coli has been characterized by X-ray absorption spectroscopy. In the resting state of the enzyme at pH 7.0, the iron is five-coordinate with an average metal-ligand bond length of 1.98 A. Binding of azide causes a reduction in the intensity of the bound state 1s-->3d transition and an increase of 0.08 A in average bond length. Both are indicative of an increase in the iron coordination number. Raising the pH from 7.0 to 10.5 causes a similar 0.08 A increase in the average bond length, again suggesting an increase in the iron coordination number. At intermediate pH (9.4), the average bond length is 2.03 A, consistent with an approximately 50:50 mixture of the limiting high and low pH forms. Similarly, the absorption edge structure varies continuously from pH 7 to 10.5. These spectra can be fit to a titration curve with a pKa of approximately 9.8. These data suggest that the pH-dependent transition, previously identified by UV-vis, EPR, and activity measurements, may be the conversion of the iron from five- to six-coordinate, presumably through coordination by hydroxide. The 1s-->3d transition for ferric superoxide dismutase at high pH is broader but not significantly less intense than that at pH 7. This suggests that the high pH form may be significantly distorted from octahedral symmetry. At pH 7, the ferric and ferric + azide samples undergo slow X-ray induced photoreduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Unliganded and cyano derivatives of cytochrome ba3 from Thermus thermophilus have been examined by UV-vis, EPR, and resonance Raman spectroscopies. Species of cytochrome ba3 investigated include its resting, as-isolated, fully oxidized state, the fully reduced, unliganded enzyme, the one-electron-reduced cyano complex, the three-electron-reduced cyano complex, and the fully reduced cyano complex. Results are compared to those obtained from similar adducts of bovine cytochrome aa3, in particular, the fully reduced cyano complex. Our objective was to identify structural similarities and differences at the ligand-binding binuclear site of the two enzymes. We observed that the inner core skeletal vibrations of cytochrome a3 are the same for similar adducts of the bacterial ba3 and mammalian aa3, indicating similar spin and iron-porphyrin coordination properties resulting in comparable porphyrin core geometries. On the other hand, many of the vibrational frequencies associated with the formyl and vinyl peripheral substituents, and the outer pyrrole carbon atoms differ between the bovine and bacterial enzymes. Use of 57Fe labeled ba3 allows identification of two separate vFe-N(His) frequencies displayed by the fully reduced, unliganded cytochrome. These frequencies, occurring at 193 and 209 cm-1, are ascribed to distinct protein conformers, which are best evidenced by the Fe-N(His) vibrations. This result is again in contrast to the bovine enzyme which has been shown by others to display a single Fe-N(His) stretching frequency at 214 cm-1. The low-frequency Fea3(2+)-CN- vibrations of the three-electron and fully reduced cyano complexes of cytochrome ba3 are identified by using 15N and 13C isotopomers of CN-. These spectral signatures are identical to those reported earlier for the one-electron-reduced cyanide adduct (cytochrome a3 reduced), showing that the Fea3(2+)-CN- vibrational frequencies are independent of the redox states of the other three metal centers. Similarly, the CuB2+ EPR signatures appear similar in both the one-electron- and three-electron-reduced cyanide adducts. On the other hand, the electronic absorption spectra of ferrous alpha 3-CN- show systematic red-shifts of the alpha band as each of the other metal centers is reduced, and other, more subtle, differences in the electronic absorptions of the three-electron-reduced and four-electron-reduced cyanide adducts are revealed in the difference spectra. The relevance of these findings toward explaining the different cyanide binding and redox chemistry described herein and toward establishing the extent of structural analogy between the oxygen binding sites of the two proteins is discussed.

We have prepared the following cadmium-113-substituted derivatives of bovine superoxide dismutase and recorded the nuclear magnetic resonance (NMR) spectrum of the cadmium: 2Cd(II), in which Cd(II) is presumed to bind to the Zn(II) site and the copper site is unoccupied, and 2Cd(II)--2Cu(I), which is analogous to the reduced form of the native protein. NMR transitions were observed at 310 ppm downfield from Cd(ClO4)2 for 2Cd(II) and at 320 ppm for the 2Cd(II)--2Cu(I)-containing proteins. In each case the observed line width was 27 +/- 2 Hz. The following conclusions were drawn. (a) The very small chemical-shift difference between the two derivatives indicates that the Cd(II) binding site is very similar in both samples. It follows from this result and previous work that the imidazolato bridge is protonated on the Cu side upon reduction of the Cu ion from the II to I valence state. (b) The extremely narrow line width of the resonance in both forms suggests a virtual identity of Cd(II) bound to both subunits of the molecule. (c) The relaxation time, T1 = 1.2 s, is caused by approximately equal contributions from chemical-shift anisotropy and dipolar interactions with nearby protons.