Azide Binding Research Articles

Understanding the Peculiarities of Azide and Thiocyanate Binding in Proteins: Use of the Small Molecule Structural Data

The complexation and supramolecular recognition of two polyfunctional anions, thiocyanate and azide, have been analysed using the X-ray structural data retrieved from the Brookhaven Protein Database (for macromolecules) and the Cambridge Structural Database (for small molecule structures). The following structural aspects have been considered: - hydrogen-bond acceptor function of the anions; - analysis of the coordination features of anions with the metals; - influence of metal complexation on the H-donor accepting properties of the anions. Lastly, a comparison of the modes of binding of the two anionic substrates was carried out. Their extraordinary activity as hydrogen-bond acceptors allows these anions to serve as a strong bridge between different molecular fragments. The coordination of thiocyanate or azide anion by the metal atoms in coordination compounds gives rise to a redistribution of anionic charge in both entities which in turn, controls the hydrogen-bonding acceptor properties of the terminal atom of the anion. The results presented in this paper provide structural information of the role of anion binding in proteins and to our knowledge, afford the first study of the binding behaviour of thiocyanate and azide in systematic manner.

The ferric form of the three human embryonic hemoglobins and their reactions with azide ions

The ferric forms of the three human embryonic hemoglobins exhibit pH titration’s with p K a values in the range 8.25–8.6, which correlate with the reduced proteins affinity for oxygen. Azide ions bind to each ferric protein in a process consisting of two separate binding steps. The equilibrium constants for this process are in the 10–30 μM range and can be assigned to the individual chain within the proteins as reactivity appears independent of the nature of the partner chain. The kinetics of the azide binding reactions occur on the second time scale at mM ligand concentrations and also indicate the presence of two distinct processes that have been assigned to each of the two chains within the proteins. Non of these processes indicate any evidence of heme–heme interactions within the ferric form of the proteins. These findings are discussed in comparison with previously reported properties of adult ferric hemoglobin.

Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding.

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, mu = 0.10 M, 25.0 +/- 0.1 degrees C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 +/- 0.3 microM, 19 +/- 1 microM and 11 +/- 1 microM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 +/- 1 mM, 50 +/- 2 mM and 12 +/- 1 mM, and for binding of pyridine were 260 +/- 50 microM, 4.5 +/- 0.5 microM and 66 +/- 8 microM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 +/- 0.1 and 6.7 +/- 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 +/- 2 mV (pH 5.40, 25.0 degrees C, mu = 0.10 M) and - 65 +/- 2 mV (pH 5.42, 25.0 degrees C, mu = 0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.

Engineering His(E7) Affects the Control of Heme Reactivity in Aplysia limacina Myoglobin

Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 Å crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.

Investigations of the myoglobin cavity mutant H93G with unnatural imidazole proximal ligands as a modular peroxide O-O bond cleavage model system.

A general inability to elucidate extensive variations in the electronic characteristics of proximal heme iron ligands in heme proteins has hampered efforts to obtain a clear understanding of the role of the proximal heme iron ligand in the activation of oxygen and peroxide. The disadvantage of the frequently applied site-directed mutagenesis technique is that it is limited by the range of natural ligands available within the genetic code. The myoglobin cavity mutant H93G [Barrick, D. (1994) Biochemistry 33, 6546-6554] has its proximal histidine ligand replaced with glycine, a mutation which leaves an open cavity capable of accommodating a variety of unnatural potential proximal ligands. We have carried out investigations of the effect of changing the electron donor characteristics of a variety of substituted imidazole proximal ligands on the rate of formation of myoglobin compound II and identified a correlation between the substituted imidazole N-3 pK(a) (which provides a measure of the electron donor ability of N-3) and the apparent rate of formation of compound II. A similar rate dependence correlation is not observed upon binding of azide. This finding indicates that O-O bond cleavage and not the preceding peroxide binding step is being influenced by the electron donor characteristics of the substituted imidazole ligands. The proximal ligand effects are clearly visible, but their overall magnitude is quite low (1.7-fold increase in the O-O bond cleavage rate per pK(a) unit). This appears to provide support for recent commentaries which concluded that the partial ionization of the proximal histidine ligand in typical heme peroxidases may not be enough of an influence to provide a mechanistically critical push effect [Poulos, T. L. (1996) JBIC, J. Biol. Inorg. Chem. 1, 356-359]. Further attempts were made to define the mechanism of the influence of N-3 pK(a) on O-O bond cleavage by using peracetic acid and cumene hydroperoxide as mechanistic probes. The observation of heme destruction in these reactions indicates that displacement of the proximal imidazole ligands by peracetic acid or cumene hydroperoxide has occurred. A combination mutation (H64D/H93G) was prepared with the objective of observing compound I of H64D/H93G with substituted imidazoles as proximal ligands upon reaction with H(2)O(2). This double mutant was found to simultaneously bind imidazole to both axial positions, an arrangement which prevents a reaction with H(2)O(2).

Effects of azide on the S(2) state EPR signals from Photosystem II.

The anion azide, N(3) (-), has been previously found to be an inhibitor of oxygen evolution by Photosystem II (PS II) of higher plants. With respect to chloride activation, azide acts primarily as a competitive inhibitor but uncompetitive inhibition also occurs [Haddy A, Hatchell JA, Kimel RA and Thomas R (1999) Biochemistry 38: 6104-6110]. In this study, the effects of azide on PS II-enriched thylakoid membranes were characterized by electron paramagnetic resonance (EPR) spectroscopy. Azide showed two distinguishable effects on the S(2) state EPR signals. In the presence of chloride, which prevented competitive binding, azide suppressed the formation of the multiline and g = 4.1 signals concurrently, indicating that the normal S(2) state was not reached. Signal suppression showed an azide concentration dependence that correlated with the fraction of PS II centers calculated to bind azide at the uncompetitive site, based on the previously determined inhibition constant. No evidence was found for an effect of azide on the Fe(II)Q(A) (-) signals at the concentrations used. This result is consistent with placement of the uncompetitive site on the donor side of PS II as suggested in the previous study. In chloride-depleted PS II-enriched membranes azide and fluoride showed similar effects on the S(2) state EPR signals, including a notable increase and narrowing of the g = 4.1 signal. Comparable effects of other anions have been described previously and apparently take place through the chloride-competitive site. The two azide binding sites described here correlate with the results of other studies of Lewis base inhibitors.

Azide binding to the trinuclear copper center in laccase and ascorbate oxidase

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1:1 to 1:7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximately 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximately 1.97 signal, with similar saturation characteristics was found in the AO : azide (1:7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti-ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1:7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g parallel region that are attributed to a perturbed T2 in protein molecules where the anti-ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6-90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.

The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies.

The effect of multiple binding of azide, N3-, on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an approximately 100-fold enhancement of the kcat/Km parameter. Chloride at physiological concentration proved to behave very similarly to N3- bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611-617], but it also enhanced some 60-fold the kcat/Km value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N3- or Cl-. At higher N3- concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N3-/CP ratios. The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.

Light scattering, CD, and ligand binding studies of ferrihemoglobin-polyelectrolyte complexes

Quasi-elastic light scattering (QELS), electrophoretic light scattering (ELS), CD spectroscopy, and azide binding titrations were used to study the complexation at pH 6.8 between ferrihemoglobin and three polyelectrolytes that varied in charge density and sign. Both QELS and ELS show that the structure of the soluble complex formed between ferrihemoglobin and poly(diallyldimethylammonium chloride) [PDADMAC] varies with protein concentration. At fixed 1.0 mg/mL polyelectrolyte concentration, protein addition increases complex size and decreases complex mobility in a tightly correlated manner. At 1.0 mg/mL of greater protein concentration, a stable complex is formed between one polyelectrolyte chain and many protein molecules (i.e., an intrapolymer complex) with apparent diameter approximately 2.5 times that of the protein-free polyelectrolyte. Under conditions of excess polyelectrolyte, each of the three ferrihemoglobin-polyelectrolyte solutions exhibits a single diffusion mode in QELS, which indicates that all protein molecules are complexed. CD spectra suggest little or no structural disruption of ferrihemoglobin upon complexation. Azide binding to the ferrihemoglobin-poly(2-acrylamide-2-methylpropanesulfonate) [PAMPS] complex is substantially altered relative to the polyelectrolyte-free protein, but minimal change in induced by complexation with an AMPS-based copolymer of reduced linear charge density. The change in azide binding induced by PDADMAC is intermediate between that of PAMPS and its copolymer.

Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: A thermodynamic, kinetic and X-ray crystallographic study

Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole binding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitreoscilla Hb) have been determined at pH 6.4 and 7.0, and 20.0 °C, in solution and in the crystalline state. Moreover, the three-dimensional structures of the diligated thiocyanate and imidazole derivatives of recombinant ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1.8 Å (Rfactor = 19.9 %) and 2.1 Å (Rfactor = 23.8 %) resolution, respectively. Ferric Vitreoscilla Hb displays an anticooperative ligand binding behaviour in solution. This very unusual feature can only be accounted for by assuming ligand-linked conformational changes in the monoligated species, which lead to the observed 300-fold decrease in the affinity of cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitreoscilla Hb with respect to that of the fully unligated homodimer. In the crystalline state, thermodynamics for azide and imidazole binding to ferric Vitreoscilla Hb may be described as a simple process with an overall ligand affinity for the homodimer corresponding to that for diligation in solution. These data suggest that the ligand-free homodimer, observed in the crystalline state, is constrained in a low affinity conformation whose ligand binding properties closely resemble those of the monoligated species in solution. From the kinetic viewpoint, anticooperativity is reflected by the 300-fold decrease of the second-order rate constant for cyanide and imidazole binding to the monoligated ferric Vitreoscilla Hb with respect to that for ligand association to the ligand-free homodimer in solution. On the other hand, values of the first-order rate constant for cyanide and imidazole dissociation from the diligated and monoligated derivatives of ferric Vitreoscilla Hb in solution are closely similar. As a whole, ligand binding and structural properties of ferric Vitreoscilla Hb appear to be unique among all Hbs investigated to date.

Resonance Raman and Fourier Transform Infrared Detection of Azide Binding to the Binuclear Center of Cytochrome bo3 Oxidase from Escherichia coli

Resonance Raman and FTIR spectra are reported for the oxidized azide-bound derivative of the quinol cytochrome bo3 oxidase from Escherichia coli. The resonance Raman spectra display three isotope-dependent vibrational modes at 419, 2040, and 2061 cm-1. The FTIR spectra display two isotope-dependent bands at 2040 and 2061 cm-1. We assign the band at 419 cm-1 to ν(Fe−N3−CuB) and the bands at 2040 and 2061 cm-1 to νas(N3). The observation of two νas(N3) modes suggests that the azide ion binds to two different enzyme conformations, both forming bridging complexes with the binuclear center. Comparison of the FTIR data of the azide-bound cytochrome bo3 and cytochrome aa3 complexes reveal that there are quantitative differences in the structure of the heme o3−CuB and heme a3−CuB binuclear pockets upon azide binding. The present data on the vibrational frequencies of the azide-bound cytochrome bo3 complex do not support the recent proposal that azide binds as a terminal ligand to CuB (Little, R. H.; Cheesman, M. ...

Azide as a competitor of chloride in oxygen evolution by Photosystem II.

Oxygen evolution by higher plants requires chloride, which binds to a site associated with the oxygen-evolving complex of photosystem II (PSII). In this study, the inhibitory effect of the anion azide was characterized using steady state measurements of oxygen evolution activity in PSII-enriched thylakoid membranes. N3- (7.8 mM) inhibited O2 evolution activity by 50% when a standard buffer containing chloride was used. By considering Cl- as the substrate in O2 evolution assays, we found azide to be primarily competitive with Cl- with an inhibitor dissociation constant Ki of about 0.6 mM. An uncompetitive component with a Ki ' of 11 mM was also found. Removal of the 17 and 23 kDa polypeptides resulted in a decrease in each inhibition constant. A pH dependence study of O2 evolution activity showed that the pH maximum became narrower and shifted to a higher pH in the presence of azide. Analysis of the data indicated that an acidic residue defined the low side of the pH maximum with an apparent pKa of 6.7 in the presence of azide compared with 5.5 for the control. A basic residue was also affected, exhibiting an apparent pKa of 7.1 compared with a value of 7.6 for the control. This result can be explained by a simple model in which azide binding to the chloride site moves negative charge of the anion away from the basic residue and toward the acidic residue relative to chloride. As a competitor of chloride, azide may provide an interesting probe of the oxygen-evolving complex in future studies.

Fourier Transform Infrared and Resonance Raman Studies of the Interaction of Azide with Cytochrome c Oxidase from Paracoccus denitrificans

The interaction of azide with oxidized cytochrome c oxidase from Paracoccus denitrificans has been studied by resonance Raman, Fourier transform infrared, and UV−vis spectroscopy. Azide binds in two phases: a high-affinity phase (Kd = 4.1 μM) in which it is bound to a nonmetal site near the binuclear center and a low-affinity phase (Kd = 11.4 mM) in which it is bound as a bridge to the binuclear center. The resonance Raman spectra of the low-affinity phase display one isotope-dependent vibrational mode at 417 cm-1. The FTIR spectra display two isotope-dependent bands at 2038 and 2056 cm-1. We assign the band at 417 cm-1 to ν(Fe−N3−CuB) and the bands at 2038 and 2056 cm-1 to νas(N3). We observe similar FTIR spectra for the azide complex of bovine heart oxidase and conclude that the binuclear center in this oxidase behaves in a manner analogous to the P. denitrificans enzyme. In contrast to mammalian cytochrome c oxidase (Li, W.; Palmer, G. Biochemistry 1993, 322, 1833−1843), the low-affinity phase observed in the interaction of azide with the P. denitrificans enzyme is not associated with binding of azide to heme a. The observation of two FTIR νas(N3) modes suggests that the azide ion binds to two different enzyme conformations, both forming bridging complexes with the binuclear center. Comparison of the UV−vis, resonance Raman, and FTIR data of the azide-bound cytochrome c aa3 from P. denitrificans and those of azide-bound quinol cytochrome bo3 suggest significant alterations in the interaction of azide with the oxidized forms of these bacterial oxidases resulting from specific structural differences within their respective heme a3−CuB and heme o3−CuB binuclear pockets.

The Crystal Structure of an Azide Complex of the Diferrous R2 Subunit of Ribonucleotide Reductase Displays a Novel Carboxylate Shift with Important Mechanistic Implications for Diiron-Catalyzed Oxygen Activation

The dinuclear Fe-center in the R2 protein of ribonucleotide reductase catalyzes oxygen activation chemistry leading to generation of the essential stable tyrosyl radical. Related oxygen reactions occur in several other diiron-containing enzyme systems where highly oxidative reaction intermediates are required to activate the substrates. Two such examples are methane monooxygenase and Δ9 stearoyl-acyl carrier protein desaturase where oxygen activation takes place at Fe-centers whose structures are similar to the Fe-center in R2. In an attempt to structurally characterize the nature of the dioxygen cleavage reaction performed by these proteins we have determined the crystal structures of two different forms of the diferrous R2 protein in the presence of azide, a potential Fe ligand. In crystals of the wt protein no azide binding was detected. The mutant protein F208A/Y122F has a larger hydrophobic pocket around the Fe-center and in the structure of this protein azide bind as an η1-terminal ligand to Fe2, the Fe ion farthest away from the tyrosine residue to be oxidized in the radical generation reaction. Glu 238, the Fe ligand most exposed into the hydrophobic pocket, coordinates the Fe-center in a novel μ-(η2,η1) bridging mode with one of the carboxylate oxygen atoms forming a bridge between the two iron ions and the other oxygen being coordinated to Fe2. Through this bridging the Fe−Fe distance is shortened to about 3.4 Å as compared to 3.9 Å for the structure of the reduced wt protein. On the basis of the novel carboxylate shift and recent data on the spectroscopic properties of the key intermediate X we propose a unique structure for intermediate X and a detailed mechanism for dioxygen cleavage. This mechanism suggests an asymmetric oxygen cleavage with a terminal oxo/hydroxo group as the major species responsible for substrate activation.

Metmyoglobin/Azide: The Effect of Heme-Linked Ionizations on the Rate of Complex Formation

The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN3, binds to metmyoglobin with a rate constant of (3.8 ± 1.0) × 105M−1s−1. Protonation of a group with an apparent pKaof 4.0 ± 0.3 increases the rate of HN3binding 6.5-fold to (2.5 ± 0.8) × 106M−1s−1. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N−3, binds to metmyoglobin with a rate constant of (4.7 ± 0.3) × 103M−1s−1, about two orders of magnitude slower than HN3. Conversion of aquometmyoglobin to hydroxymetmyoglobin slows azide binding significantly. Binding of HN3to hydroxymetmyoglobin cannot be detected, while N−3binds to hydroxymetmyoglobin with a rate of 5.7 ± 3.2 M−1s−1, almost three orders of magnitude slower than N−3binding to aquometmyoglobin. Protonation of the distal histidine facilitates HN3dissociation from the complex. HN3dissociates from the metmyoglobin/azide complex with a rate constant of 18 ± 6 s−1, while the azide anion dissociates with a rate constant of 0.16 ± 0.02 s−1, about 100 times slower. The apparent pKafor His-64 is essentially the same in metmyoglobin and the metmyoglobin/azide complex, 4.0 ± 0.3 and 4.4 ± 0.2, respectively. The ionic strength dependence of the observed association rate constant is influenced by both primary and secondary kinetic salt effects. The primary kinetic salt effect is anomalous, with the rate of N−3binding decreasing with increasing ionic strength above the isoelectric point of metmyoglobin where the protein has a net negative charge. The ionic strength dependence of the dissociation rate constant can be described solely in terms of the ionic strength dependence of the acid dissociation constant for His-64 in the metmyoglobin/azide complex, a secondary kinetic salt effect.

A structure-based mechanism for copper-zinc superoxide dismutase.

A reaction cycle is proposed for the mechanism of copper-zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle. This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations. The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant. Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9%. Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following: (i) The protein backbones (the "SOD rack") remain essentially unchanged. (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 A from its Cu(I) position in the trigonal plane formed by His46, His48, and His120. The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal. The copper geometry remains three coordinate, but the His48-Cu bond distance increases by 0.18 A. (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 A from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character. The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms. The His48-Cu distance increases by 0.70 A, suggesting that His48 becomes an axial ligand. (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding. Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 A, respectively, in these structures. (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand. Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.

A Thermodynamic Study of Azide Binding to Myoglobin

The visible absorption spectrum associated with the heme protein myoglobin changes as a result of azide binding to the iron center. Therefore, a titration with azide can be monitored and concentrations of the myoglobin-azide adduct quantified. From these ligand binding data, the dissociation constant and number of binding sites can be determined using the Scatchard method for analysis. This laboratory project can be completed in two 3-hour lab periods. Evaluation of other thermodynamic properties is possible by exploring the temperature dependence of these binding data. A third lab period is required for students to probe the temperature dependence.

Mechanistic Studies of the Zirconium−Triisopropanolamine-Catalyzed Enantioselective Addition of Azide to Cyclohexene Oxide

The mechanism of the enantioselective ring-opening of cyclohexene oxide by Me3SiN3, catalyzed by zirconium complexes of the C3-symmetric ligand (+)-(S,S,S)-triisopropanolamine, has been investigated. Measurements of molecular weights of precatalyst species show that complexes are formed with average trimeric aggregation. Kinetics measurements reveal the overall process to be approximately half order in total zirconium, epoxide, and Me3SiN3 components. The reaction also shows a strong nonlinear relationship between enantiomeric excess of product azido ether vs the incorporation of (R,S,S)-triisopropanolamine ligand in the catalyst mixture. On the basis of these and other results, a preequilibrium interconversion of dimeric and tetrameric zirconium−triisopropanolamine species is proposed to occur rapidly with respect to the rate of epoxide ring-opening, with the dimeric form being the active catalyst. The reaction is accelerated by silyl ethers or by small amounts of water or alcohol, whereas larger amounts of protic additives inhibit the reaction. Enantioselectivity is eroded at catalyst concentrations less than 1 mole-percent and at high concentrations of cyclohexene oxide. Both enantioselectivity and rate are influenced to a small extent by the nature of the silyl azide employed for the first catalytic turnover, suggesting that a silyl fragment becomes irreversibly incorporated in the catalyst structure. It is proposed that catalytic activity requires the cooperative action of two zirconium centers for the binding and delivery of azide to epoxide.

Two-Dimensional ESEEM Spectroscopy of Nitrogen Hyperfine Couplings in Methemerythrin and Azidomethemerythrin

The application of a two-dimensional ESEEM spectroscopy, known as HYSCORE, to the study of the hyperfine couplings from the nitrogens of histidine and azide ligands in mixed-valent states of methemerythrin and azidomethemerythrin produced by low-temperature (77 K) γ-irradiation is described. The HYSCORE spectra contain groups of well-separated cross-peaks from directly coordinated and remote histidine nitrogens, and from nitrogens of the azide ligand. The observation of cross-features correlating double-quantum transitions allows direct estimation of the hyperfine couplings from all coordinated histidine nitrogens of semimethemerythrin which vary between 5 and 15 MHz as well as their changes upon azide binding. The results obtained show that two-dimensional spectroscopy provides significantly more information compared to one-dimensional techniques for the characterization of the paramagnetic centers involving large numbers of magnetically nonequivalent nuclei. This work forms the basis for the application...

Magnetic studies of the trinuclear center in laccase and ascorbate oxidase approached by EPR spectroscopy and magnetic susceptibility measurements

The trinuclear centers in Rhus vernicifera laccase and Cucumis sativus ascorbate oxidase have been studied by EPR spectroscopy and magnetic susceptibility measurements over the wide range of 5 K to 300 K. The EPR spectra showed that type II copper receives increasing tetrahedral distortion with raising temperature. Magnetic susceptibilities of laccase showed that both of type I and type II coppers are almost fully paramagnetic since the antiferromagnetic interaction between type III coppers is extremely strong from 5 K to 300 K. On the other hand, the effective magnetic moment of ascorbate oxidase is contributed by ca. 1.7 Cu 2+ even below ca. 100 K, since type II Cu is partly in the reduced form. The effective magnetic moment continuously increased with raising temperature because the antiferromagnetic interaction between type III coppers is not as strong as in the case of laccase. The simulation of the SQUID measurement results suggested that the conformational change of the ascorbate oxidase molecule caused the temperature dependence of the antiferromagnetic interaction. The type II Cu EPR signals in laccase and ascorbate oxidase were conspicuously broadened with raising temperature because of the increasing contribution of the triplet state by type III Cu's and/or of the rapid relaxation which finally led to only ca. 30% detection of the type II Cu signals at room temperature. The stepwise binding of azide to the trinuclear center made one of type III Cu's to be EPR detectable. SQUID measurements indicated that only one Cu in the trinuclear center is paramagnetic and other two Cu's are antiferromagnetically coupled for both of the one- and two-azide bound forms. The binding mode of azide to the trinuclear center was discussed based on some models.