Activation of Soluble Guanylyl Cyclase by Four-Coordinate Metalloporphyrins: Evidence for a Role for Porphyrin Conformation ² Heather S. Carr, ‡ Dat Tran, § Mark F. Reynolds, ‡ Judith N. Burstyn,* ,‡ and Thomas G. Spiro* ,§ Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue, Madison, Wisconsin 53706, and Hoyt Laboratory, Princeton UniVersity, Washington Road, Princeton, New Jersey 08544 ReceiVed April 17, 2001; ReVised Manuscript ReceiVed June 22, 2001 ABSTRACT: Four-coordinate metalloporphyrins activate soluble guanylyl cyclase. Ni II PPIX and Cu II PPIX are high affinity activators, with activation constants of 24 and 17 nM, respectively. Both metalloporphyrins remain stably bound to the enzyme, enabling spectroscopic characterization of the Ni(II)- and Cu(II)- reconstituted protein. Electronic absorption and resonance Raman spectroscopy reveal that Ni II PPIX remains four coordinate when bound to soluble guanylyl cyclase. Analysis of the vibrational frequencies of the Ni(II)-reconstituted enzyme suggests that the protein imposes a constraining force on the porphyrin, favoring a planar conformation. Spectroscopic data for the Cu(II)-substituted protein are also consistent with four coordination. The intensification of the vibrational modes of the peripheral vinyl groups in both Ni(II)- and Cu(II)-reconstituted soluble guanylyl cyclase are consistent with a substantial influence of the protein on the porphyrin environment. Together these data support a model where activation of soluble guanylyl cyclase correlates with the absence of a metal-to-proximal histidine bond and with decreased conformational freedom for the tetrapyrrole in the activated state. Nitric oxide (NO) is an important physiological signaling molecule, and a complete understanding of how NO regulates it primary target, soluble guanylyl cyclase, has yet to be achieved. Soluble guanylyl cyclase (sGC) 1 is a heterodimeric heme protein that catalyzes the transformation of GTP to cGMP, a key intracellular second messenger (1). Although the b-type heme (Fe II PPIX) is not required for sGC to exhibit basal catalytic activity, it is essential for NO-induced activation (2). The sGC heme in its resting state is either 5- or 6-coordinate, with histidine(s) as the axial ligand (3-6). NO coordinates to the heme cofactor, breaking the iron- histidine bond(s) to form a five-coordinate species (3, 6, 7). It is proposed that the scission of the metal-histidine bond mediates a conformational change that leads to the activation of the enzyme (8); however, the details of this process are unknown. Tetrapyrroles other than NO-heme b influence the activity of sGC. The free base of heme b, protoporphyrin IX (PPIX), activates sGC, as do mesoporphyrin and hematoporphyrin (9, 10). All of these tetrapyrroles have propionate substituents at the 6 and 7 positions, while the substituents at the 2 and 4 positions are vinyl and ethyl or hydroxyethyl, respectively. Other alterations of the propionate or vinyl substituents render the tetrapyrroles inhibitory or ineffective as activators (9). Metalloporphyrins other than heme b also affect sGC. ZnPPIX, CoPPIX, and MnPPIX all inhibit basal sGC activity (8, 9, 11). Only CoPPIX and FePPIX activate sGC in the presence of NO, an observation that has been correlated with the formation of a five-coordinate nitrosyl adduct (8). Kinetic analysis reveals that PPIX and NO-heme activate sGC by similar mechanisms, implying that other porphyrins may influence the protein in the same manner (2). Activation of sGC by non-native porphyrins and metalloporphyrins may therefore provide insight into the manner in which the activated conformation is stabilized. As part of our ongoing studies of the mechanism of activation of sGC, we have investigated the effects of four- coordinate metalloporphyrins on the activity of sGC. The data described above suggest that the presence of the heme b periphery and the absence of a metal histidine bond are necessary for activation of sGC. To test this hypothesis, the effects of air-stable Ni II PPIX and Cu II PPIX, electron-rich porphyrins that should remain four-coordinate, were studied by kinetic and spectroscopic techniques. As described herein, both metalloporphyrins are activators and they are 4-coor- dinate when bound to sGC. The data presented suggest that not only is the peripheral substitution pattern important in sGC activation, but the conformation of the tetrapyrrole is also of consequence. EXPERIMENTAL METHODS Materials. Bovine lung was obtained from Schroedl’s Meat Market in Jefferson, WI. The anti-BSA-agarose resin was ² This work was supported in part by NIH Grants GM-33576 (T.G.S.) and HL-65217 (J.N.B). NIH fellowship support is gratefully acknowl- edged (GM-18949, D.T.; GM-08505, H.S.C.; GM-08293, M.F.R.). * To whom correspondence should be addressed. J.N.B.: phone (608) 262-0328; fax (608) 262-6143; E-mail burstyn@ chem.wisc.edu. T.G.S.: phone (609) 258-3907; fax (609) 258-0348; E-mail spiro@chemvax.princeton.edu. ‡ University of WisconsinsMadison. § Princeton University. 1 Abbreviations: sGC, soluble guanylyl cyclase; PPIX, protopor- phyrin IX; TEA, triethanolamine; DTT, D,L-dithiothreitol; SNAP, S-nitroso-N-acetyl penicillamine; IBMX, isobutylmethylxanthine; DMF, dimethyl formamide; DMSO, dimethyl sulfoxide; µBCA, micro bicin- choninic acid protein assay; RR, resonance Raman; pip, piperidine; CTAB, cetyl trimethylammonium bromide; cyt c; cytochrome c; MesoP, mesoporphyrin; MP-11, microperoxidase 11, an 11-amino acid heme bearing peptide derived from cyt c; P, porphine; MES, 2-(N-morpholi- no)ethanesulfonic acid; P i, phosphate buffer. 10149 Biochemistry 2002, 41, 10149-10157 10.1021/bi010777k CCC: $22.00 © 2002 American Chemical Society Published on Web 07/12/2002