4338 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Inorg. zyxwvutsr Chem. 1994, 33, zyxwvu 4338-4343 lH-13C HETCOR Investigations on Heme-Containing Systems Lucia Banci, Ivano Bertini,' Roberta Pierattelli, and Alejandro J. Vilat Department of Chemistry, University of Florence, 50121 Florence, Italy Received January 6, zyxwvuts 1994@ IH- 13CHETCOR studies were performed on some low-spin heme-containing systems. From the available data or our extended proton assignment, complete carbon assignments were performed. The carbon (and proton, when not yet already performed) chemical shifts were factored out into metal-centered pseudocontact shifts and contact (plus ligand-centered pseudocontact) shifts. The ratios between proton and carbon contact shifts were found to be constant for the symmetric bis(imidazo1e) protoporphyrin and to vary for the asymmetric protein systems. This feature is discussed also in the light of the data already available on other heme proteins. Introduction The improvement of NMR technologies permits the obtain- ment of further information on systems already largely inves- tigated. 'H reverse detection in heteronuclear correlation (HETCOR) spectroscopy of paramagnetic compounds may provide scalar hetero correlation even for broad signals and under natural-abundance conditions for the heteronu~leus.'-~ Here we present investigations of bis(imidazo1e)-hemin (PPImz), metmyoglobin cyanide (MbCN), and cytochrome b5 (Cytbs). All of these systems contain low-spin iron(II1). PPIm2 represents a good model for the detection of all the expected cross peaks because concentrated solutions can be obtained. Indeed, lH-13C NMR cross peaks are observed even for the fully bound imidazole nuclei which have the broadest proton lines (230 Hz). Reverse detection HETCOR studies are already available, with 13C in natural abundance, for cytochromes c2 and zyxwvutsrqp c551' and oxidized high-potential iron sulfur proteins (H~PIPs).~ A IH-I3C HETCOR direct study is available for MbCN? which allows us to establish the superiority of reverse detection even in the case of fast-relaxing systems. The assignment of the hyperfine-shifted proton signals is available for the three systems,s-10 whereas the 13C resonance assignment is known for some signals of MbCN4 and for the bis(imidazo1e) adduct of a synthetic porphyrin." No I3C spectrum is available for Cytb5. Actually, rat Cytbs is present in two isomers differing in the heme orientation inside the protein frame;I2 both isomers are investigated here. The zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA + Present address: Dto. Qca Biologica, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina. @Abstract published in Advance ACS Abstracts, July 15, 1994. (1) Timkovich, R. Inorg. Chem. 1991, 30, 37. (2) Santos, H.; Tumer, D. L. Eur. J. Biochem. 1992, 206, 721. (3) Bertini, I.; Capozzi, F.; Luchmat, C.; Piccioli, M.; Vila, A. J. J. Am. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (4) Yamamoto, Y. FEBS Lett. 1987, 222, 115. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (5) McLachlan, S. J.; La Mar, G. N.; Sletten, E. zyxwvuts J. Am. Chem. SOC. 1986, (6) Yamamoto, Y.; Osawa, A,; Inoue, Y.; Chujo, R.; Suzuki, T. FEBS (7) Emerson, S. D.; La Mar, G. N. Biochemistry 1990, 29, 1545. (8) Lee, K.-B.; La Mar, G. N.; Kehres, L. A,; Fujinari, E. M.; Smith, K. M.; Pochapsky, T. C.; Sligar, S. G. Biochemistry 1990, 29, 9623. (9) Yu, L. P.; La Mar, G. N.; Rajarathnam, K. J. Am. Chem. SOC. 1990, 112, 9527. (10) Lee, K.-B.; La Mar, G. N.; Pandey, R. K.; Rezzano, I. N.; Mansfield, K. E.; Smith, K. M.; Pochapsky, T. C.; Sligar, S. G. Biochemistry 1991, 30, 1878. Chem. SOC. 1994, 116, 651. 108, 1285. Lett. 1989, 247, 263. (11) Goff, H. M. J.Am. Chem. Sac. 1981, 103, 3714. (12) Rogers, K. K.; Pochapsky, T.C.; Sligar, S. G. Science 1988,240, 1657. 0020- 1669/94/1333-4338$04.50/0 magnetic anisotropy tensor, which provides the metal-centered pseudocontact shifts, is available for MbCN13 and for the two isomers of C ~ t b 5 . l ~ Once the carbon signals of the heme substituents are assigned, we examine the problem of a possible relationship between 'H and 13C hyperfine shifts. Such a problem has been investigated for p~ridine,'~ triphenylphosphine,'6 and N-oxide" ligands bound to nickel(I1) and cobalt(I1); evidence for a complex spin density transfer mechanism had been found. Recently, Turner18 addressed the problem for cytochrome c, separating the various contributions to the shifts for both 'H and 13C resonances and calculating the spin density on the resonating nuclei. He found that the hyperfine coupling constants of the proton nuclei of the methyls attached to sp2 carbons of the heme pyrrole rings are dependent on the heme position.18 We now show that such variability is a general feature and that the extent of variability is quite large. Evidently, chemical shifts in paramagnetic molecules are sufficiently sensitive to be able to monitor very subtle structural inequivalences. Materials and Methods Hemin-iron(II1) chloride was obtained from Sigma Chemical Co. and used without further purification. The bis(imidazo1e) complex was prepared by adding a4 equiv of imidazole to a solution of hemin chloride (20 mM) in deuterated dimethyl sulfoxide ((CzH3)2SO). Cytochrome bs was isolated from Escherichia coli strain TB-1, generously provided by Dr. S. G. Sligar. The protein was isolated and purified using the previously reported procedure.19 The NMR sample (8 mM) was in 100 mM phosphate buffer (pH 7.1) in D20. Sperm whale myoglobin was purchased from Sigma Chemical Co. and used without further purification. The metcyano complex was prepared by addition of small quantities of solid KCN to a 0.1 M NaCI- D20 solution of the enzyme (10 mM) at pH 7.4. The NMR spectra were recorded on a Bruker AMX 600 spectrometer equipped with a 5-mm inverse detection probe. The HMQC 'H-W spectra consisted of 128-360 experiments acquired with 1K data points each, in the magnitude mode, by using the standard pulse sequence20-22 (13) Emerson, S. D.; La Mar, G. N. Biochemistry 1990, 29, 1556. (14) Guiles, R. D.; Basus, V. J.; Sarma, S.; Malpure, S.; Fox, K. M.; Kuntz, (15) Morishima, I.; Okada, K.; Yonezawa, T.; Goto, K. Chem. Commun. (16) Doddrell, D. M.; Roberts, J. D. J. Am. Chem. SOC. 1970, 92, 6839. (17) Bertini, I.; Luchinat, C.; Scozzafava, A. Inorg. Chim. Acta 1976, 19, (18) Tumer, D. L. Eur. J. Biochem. 1993, 211, 563. (19) von Bobman, S. B.; Schulder, M. A,; Jollie, D. R.; Sligar, S. G. Proc. (20) Reynolds, J. G.; Coyle, C. L.; Holm, R. H. J. Am. Chem. SOC. 1980, I. D.; Waskell, L. Biochemistry 1993, 32, 8329. 1970, 1535. 201. Natl. Acad. Sci. U.S.A. 1986, 83, 9443. 102, 4350. 0 1994 American Chemical Society