VOLUME115, NUMBER 6 MARCH24, 1993 0 Copyright 1993 by rhc American Chemical Sociery
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Crystal Structure and Electron Spin Echo Envelope Modulation Study of [ Cu( II)(TEPA)(NO2)]PF6(TEPA = Tris[ 2 4 2-pyridy1)ethyllamine): A Model for the Purported Structure of the Nitrite Derivative of Hemocyanin Feng Jiang,*vt Rebecca R. Conry,*Luigi Bubacco,s ZoltQnT eklh,* Richard R. Jacobson,*Kenneth D. Karlin,*and Jack Peisach 0
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Contribution from the Department of Molecular Pharmacology and Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, and Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21 218. Received September 25, 1992 Abstract: The coordination properties of nitrite ion (NO2-) with copper are relevant to investigations of a variety of copper proteins, including those involved in nitrogen oxide processing. Here, we use X-ray diffraction and electron spin echo envelope modulation (ESEEM) spectroscopy to investigate the coordination and electronic structure of a nitritecopper( 11) model compound, [Cu(1l)(TEPA)NO2]PF6(TEPA = tris[Z(Zpyrid 1)ethyllamine). In the crystal (C21H2N,Cu02PF6 crystallizes in the monoclinic b = 13.124(6) A, c = 15.692(7) and /3 = 92.39(4)"), nitrite is shown space group P 2 , / c with 2 = 4, a = 11.817(8) to bind as an equatorial ligand to Cu(I1) through oxygen, rather than nitrogen. In frozen solution, the detection of ESEEM from the nitrite I4N indicates O-ligation as well. The quadrupole and electron-nuclear coupling tensors for the nitrite I4N are obtained by field-dependent ESEEM spectral simulation. The quadrupole parameters, e2qQ = 5.66 MHz and 7 = 0.31, are similar to those for diamagnetic metal nitrites. The net charge on the nitrite nitrogen calculated from the quadrupole parameters, based on Townes-Dailey theory, suggests that the O N 0 angle of bound nitrite is between 11 1 " and 116" in frozen solution, as compared to 115" in the crystal. The principal directions of the quadrupole tensor and the electron-nuclear coupling tensor relative to that of the g tensor suggest that the O N 0 orientation relative to the Cu(Il)(TEPA) structure in frozen solution is essentially the same as that in the crystal. The ability to observe modulations arising from Cu(I1)-coordinated l4NOCallows us to probe for an equatorial nitrito ligand to Cu(I1) in solution samples. An attempt was made to substantiate the claim that nitrite binds equatorially through oxygen to Cu(I1) in hemocyanin after nitrite treatment (Solomon, E. I. In Copper Proleins; Spiro, T. G . . Ed.; Wiley-Interscience: New York, 1981; pp 43-108) by searching for I4NO2-modulations in the ESEEM spectrum. None were found, suggesting that nitrite does not coordinate to the Cu(1I) of hemocyanin as claimed.
1,
Introduction'
CU-NO,~complexes are relevant in the study of copper-containing enzymes involved in the denitrification process,2 Le., the bacterial dissimilatory reduction of nitrate and nitrite to NO, N20, :To whom correspondence should be addressed. Department of Molecular Pharmacology, Albert Einstein College of Medicine of Yeshiva University. 'The Johns Hopkins University. Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University.
0002-7863/93/ 15 15-2093$04.00/0
1,
and N,. In particular, copper-nitrite complexes are potentially relevant to the nitrite reductases3 which convert NO2- to NO ( I ) Abbreviations: ESEEM, electron spin echo envelope modulation; TEPA, tris[2-(2-pyridyl)ethyl]amine;NQl, nuclear quadrupole interaction; NQR, nuclear quadrupole resonance; efg, electric field gradient; CW, continuous wave; bpy, 2,2'-bipyridyl; terpy, terpyridine; HB('Bu-pz) ,* hydrotris(3-fert-butylpyrazolyl)borate anion. (2) (a) Kroneck, M. H.; Zumft, W.G. In Denifrijicafion in Soil and Sediment; Revsbech, N. P., Ssrensen, J., Eds.; FEMS Symposium 56; Academic: London, 1990 pp 1-20. (b) Hochstein, L. 1.;Tomiinson, G. A. Annu. Reu. Microbiol. 1988, 42, 231-261. (c) Knowles, R. Microbiol. Rev. 1982, 46, 43-70. (d) Averill, B. A,; Tiedje, J. M. FEES Lett. 1982, 138. 8-12.
0 1993 American Chemical Society
Jiang et al.
2094 J. Am. Chem. Soc., Vol. 1 1 5, No. 6, 1993
and/or N 2 0 , reactions presumed to proceed by nitrite binding to Cu(II), followed by dehydration and reduction. In addition, small molecules such as nitrite have been used as spectroscopic and reactivity probes of the active sites of metalloenzymes, an example being the studies involving N O and NO; with hemocyanin; both of these reagents produce the same product, which has been proposed to be a copper(I1)-nitrite complex: although this has been que~tioned.~ The coordination of a metal ion to nitrite can, in theory, occur either through oxygen or nitrogen. Although X-ray data can be used to unequivocally make a structural assignment for the crystalline sample, structural assignment for solution samples is more difficult. One can, however, make an assignment from the nitrite-I4N quadrupole parameters, e2qQ and q, which are determined by the electron population in the valence-shell orbitah6 Such measurements have been carried out using nuclear quadrupole resonance spectroscopy, but only for solid samples. In the presence of a paramagnetic metal ion, ESEEM' spectroscopy can be applied to obtain the quadrupole parameters of metal-bound quadrupolar nuclei in solution. Under favorable conditions, it is possible to determine &qQ and q, as well as the orientation of the quadrupole tensor with respect to a structural feature, as in this study, where we have been able to make a determination of the structure of a metal-nitrite complex. In this investigation we have studied the structure of a copper(I1)-nitrite complex with a polydentate nitrogenous ligand, [cu(II)(TEPA)(No,)]PF,, both in the crystalline form by X-ray diffraction and in frozen solution by ESEEM spectroscopy. For the first time we have been able to identify the ESEEM arising from Cu(I1)-coordinated I4NO lqyyl> 1qxx1.54The quadrupole parameters are determined by eqs 7 and 8. e2qQ = e2q0Q[(1- CotZe)/
- 1 / 2 ~4- (Cot2 e - 1 / 2 ) 6 ~ 0 ]
e2qQtl = e2qoQ[%('J~o- *)I
(7) (8)
Assuming 1 is 2, the electron population for the lone-pair electrons, 1 and uNo, can then be obtained from e2qQ and q, when 0 is known. The charge on the nitrogen atom, pN, can be obtained from eq 9. PN = 3 - 26No - T (9) This was calculated for NO< in solid NaN02, where 8 q Q is 5.79 MHz, q is 0.405, and LON0 is 1 The value for uN0was calculated as 1.18 and that for 1as 1.02, which gives a net charge of -0.38 e on the nitrogen and -0.3 1 e on each of the oxygen atoms. The net charges obtained this way are in good agreement with the corresponding values of -0.36 and -0.32 e estimated from spontaneous polarization studieseS6 The net charge on the nitrite nitrogen in 1 can be calculated for a given O N 0 angle, using the quadrupole parameters obtained by ESEEM spectral simulation. In previous studies of various crystalline Cu(I1)-NO< c o m p l e x e ~ , ~the ~ - ~O~N- 0~ angle ~ for NO; was shown to vary from 1 10' to 125'. Figure 7 shows the net charge at the nitrite nitrogen calculated for this range of angles. The charges obtained the same way for solid N a N 0 2 and gaseous tram-HN02 are -0.38 and -0.03 e, respectively. They can be ~
(52) The term e'q,,Q is the nuclear quadrupole coupling constant for an "N atom with an unbalanced p electron. Depending on the total charge on the atom in question, the coupling constant varies. This value has been choscn between 8 and IO MHz4"' in NQR studies to reach an agreement with theoretical calculations. To be consistent with early works on NO2-:"'" the value of IO MHz is used in the present investigation. (53) (a) Guibe, L.; Lucken, E. A. C. Mol. Phys. 1966, IO, 273-281. (b) Shempp, E.; Bray, P. J. J . Chem. Phys. 1967, 46, 1186-1 190. (54) Several LCAO-SCF-MO calculations'' for NO,- have shown that r - I) > 0, with our assignment of the < u\(). Since q , , - q ! , = principal axis, we have 1q:J > Iq,,1 > lqu!I. The quadrupole coupling constants for frons-HNO, from a microwave study" are in fair agreement with those we obtained for our model complex, indicating that NO, and HNO? rmmble each other, at least regarding the electronic structure about their respective nitrogen nuclei. The principal axes of the quadruple coupling tensor of HNO:, which are almost coincidental with the symmetry axis of the molecule, are in accord with our assignment. (55) (a) Petrongolo. C.; Scrocco, E.; Tomasi, J. J. Chem. Phys. 1968. 48, 407-41 1. (b) Bonaccorsi, R.;Petrongolo, C.; Scrocco, E.;Tomasi. I. J. Chem. Phys. 1968, 48. 1497- 1499. (56) Kay, M. I.; Frazer, 8. C. Acfo Crysfallogr. 1961, 14, 56-57.
(57) Lucken, E. A. C. Nuclear Quadrupole Coupling Consfants; Academic: New York, 1969; pp 147-155. ( 5 8 ) Phillips, S. E. V.; Stevens, C.; Ogel. 2. B.; McPherson, M. J.; Keen, J. N.; Schoot Uiterkamp, A. J. M. FEBS Lett. 1972, 20, 93-96. (59) Van der Deen, H.; Howing, H. Biochemisfry 1977, 16, 3519-3525. (60) Verplaetse. J.; Van Tornout, P.; Defreyn, G.; Witters, R.;Lontie, R. Eur. J. Biochem. 1979, 95, 327-331. (61) Westmoreland, T.D.; Wilcox, D. E.; Baldwin, M. I.; Mims, W. 8.; Solomon, E. I. J . Am. Chem. SOC. 1989, I l l , 6106-6123.
J . Am. Chem. Soc., Vol. 115, No. 6, 1993 2101
Crystal Structure and ESEEM of fCu(II)(TEPA)(N0,)]PF6
i 0
v = 8.5 G H ~ H = 2740 G T = 257 ns
2
4
6
8
nd
-\
Frequency (MHz) Figure 8. Three-pulse ESEEM spectra of (A) the I4NO2-derivatives of hemocyanin and (B) [ C U ( I I ) ( T E P A ) ( ' ~ N O , )studied ] under similar experimental conditions. Measurement conditions: microwave frequency, (A) 8.50 GHz and (B) 8.55 GHz; magnetic field, 2740 G; T = 257 ns. The lines observed in spectrum A are attributed to multiple remote nitrogens of Cu(I1)-coordinated histidine imidazoles.
as the electron-nuclear coupling to nitrogen would undoubtedly be too weak to resolve with conventional EPR. The arguments supporting nitrite as a bridging ligand are not direct and rely heavily on an analysis of spectroscopic data!\ An independent argument for nitrite as a bridging ligand between both copper atoms at the active site is based on the sluggishness of reactivity of the nitrite derivative with azide, as compared to the same reaction for the half-met aquo derivative of the protein. Yet chemical analysis of the hemocyanin product of the nitrite reactionS showed that the amount of bound nitrite was far less than stoichiometric with the EPR detectable copper, especially for preparations produced under anaerobic conditions. Since the arguments for bridging nitrite coordination are largely inferential rather than direct, it was felt that a specific test should be made for its presence by probing for weak I4N magnetic coupling arising from a putative exogenous nitrogenous ligand to Cu(1I) using ESEEM spectroscopy. The ESEEM spectrum for the l 4 N O derivative ~ of hemocyanin is shown in Figure 8A. An identical spectrum is obtained for the preparation made with ISNOT (not shown), and both are significantly different from those obtained for [CU(II)(TEPA)(~~N~~)]PF~ (Figure 8B). For the protein, one broad and one narrow line are obtained in the lowfrequency region of the spectrum at 0.73 and 1.45 MHz, with the broader one consisting of two unresolved lines. These are characteristic I4N modulations arising from the remote nitrogen of Cu(II)-coordinated histidine imida~ole.~.'~ The presence of a sum frequency of 2.13 MHz (the sum of 0.73 and 1.45 MHz) is indicative of multiple imidazole coordination to C U ( I I ) . ~This ~ is consistent with the X-ray crystal structure of deoxy hemocyanin, where each of the copper atoms is bound to two close-lying imidazoles, with a third further away.63 The characteristic frequencies at 0.97,3.78,4.86, and 5.46 MHz and absent in the observed for [CU(II)(TEPA)(~~NO,)]PF, (Figure 8B) are not spectrum for [CU(II)(TEPA)(~~NO~)]PF, observed in nitrite-treated hemocyanin. Both the 4.86- and 5.46-MHz lines arising from nitrite in the model are well separated from the frequencies attributed to the remote nitrogen of Cu(11)-coordinated histidine imidazole in the protein. Although the 4.86-MHz line for nitrite in the model has about a quarter of the intensity of the NQI lines for the remote I4N of the coordinated imidazoles in the protein, it would clearly be observed were the (62) McCracken, J.; Pember, S.;Benkovic. S. J.; Villafranca, J. J.; Peisach, J . J . Am. Chem. SOC.1988, 110. 1069-1074. (63) Gaykema, W. P. J . ; Hol, W. G.J.; Vereijken, J . M.; Soeter, M. M.; Bal, H. J.; Beintema, J . J. Narure (London) 1984, 309. 23-29.
D
L I
0
I
(
psec
L
1
t
I
J--2
1 T
C
)
Figure 9. Two-pulse spin echo envelopes for (A) I4NO< and (B) 15N02derivatives of hemocyanin and for 1 prepared with (C) I4NO,- and (D) lSNO2-.Measurement conditions: microwave frequency, (A) 8.50 GHz, (B) 8.52 GHz, and ( C and D) 8.55 GHz; magnetic field, (A and B) 2932 G, ( C and D) 2981 G. Echo amplitudes of all four data sets were normalized to that of the I4NO2- derivative of hemocyanin.
coupling between the nitrite nitrogen and the Cu(I1) unpaired electron in the protein comparable to that in 1. The nitrite in 1 binds to Cu(I1) primarily through one oxygen, with nitrogen uncoordinated. Were the nitrite to act as a bridging ligand in the protein, with one oxygen bound to Cu(I1) and the nitrogen bound to Cu(I),6' the electron population at the lone-pair orbital would be expected to decrease so that SqQ would decrease as well. The quadrupole frequencies would then be different from those in 1. But, since the electron-nuclear coupling between the Cu(I1) unpaired electron and the I4N nucleus would likely be similar, the peak intensities in the ESEEM spectra should be comparable, about of that of the Cu(I1)-coordinated histidine imidazoles. Were nitrite to act as a bridging ligand and bind to divalent copper equatorially through oxygen, a difference would be expected in the S E E M spectra for I4NO~and ISNO~-treated hemocyanin. None was found. An alternative approach for demonstrating nitrite binding is to look for spectral differences in a twepulse experiment between I4NO
