Inorg. Chem. 1986, 25, 3734-3740
3734
Contribution from the Department of Chemistry, University of Illinois a t Chicago, Chicago, Illinois 60680
Electronic Effects on the Spectra and Carbon Monoxide Affinities of Heme and (Thiolato)heme Complexes. Cis and Trans Effects Emily M. Gaul and Richard J. Kassner* R e c e i v e d January 16, 1986
The absorption spectra of n-butanethiolate complexes of different iron porphyrins and their C O complexes were measured as models for the reported spectra of the corresponding heme-substituted cytochrome Pds0. The CO affinities of solvent and n-butanethiolate-coordinated complexes of different iron porphyrins in dimethylacetamide were also measured for comparison to reported cis effects on the CO affinities of heme-substituted cytochrome P450 as well as (imidazo1e)heme complexes. C O affinities of (n-butanethio1ato)heme complexes follow the order diacetyldeutero > spirographis > 2-acetyl-4-deutero > proto > meso, corresponding to the order of increasing porphyrin basicity. C O affinities of solvent-coordinated iron porphyrins follow the order proto > diacetyldeutero > dicyano, corresponding to the order of decreasing porphyrin basicity. The slopes of !he Brmsted type plots ( p E 0 vs. porphyrin pK3) associated with these cis effects were shown to exhibit an apparent linear dependence on the trans ligand basicity. The absorption spectra and C O affinities of protohemes and diacetyldeuterohemes coordinated to thiolates of decreasing basicity were also measured to assess the possible trans effects. CO affinities of different (thio1ato)diacetyldeuteroheme complexes followed the order 4-fluorothiophenolate > methyl thioglycolate > phenylmethanethiolate > n-butanethiolate, corresponding to the order of increasing thiolate basicity. The relationship between the spectroscopic and C O binding properties and the effective basicity of the thiolate ligand in the C O cytochrome P,,, complex is discussed.
Introduction The cytochromes P450 constitute a class of monooxygenase heme proteins t h a t catalyze the hydroxylation of a wide variety of molecules including xenobiotics and steroids in eukaryotes and camphor, the growth substrate for Pseudomonas putidu, in prokaryotes.1-2 Numerous chemical and spectroscopic studies have identified heme protein intermediates in the reaction cycle and have attempted to characterize the heme iron coordination in order to understand the biochemical mechanism i n v o l ~ e d . ~Studies .~ of carbon monoxide binding to thiolate heme models5-' h a v e indicated cysteine ligation to the heme iron in the protein. Resonance Raman studies8of ferric cytochrome P450 and EXAFS9 studies of ferric and ferrous cytochromes P450 have confirmed these and other earlier assignments. Several investigators have noted significant differences between the carbon monoxide binding properties of cytochromes P450 from different sources (bacterial, liver microsomal, adrenal cortex microsomal) I3-l8, from different isozymes (phenobarbitol-induced P450 LM-2 vs. benzoflavone-induced P450 LM-4),16 and between cytochromes P450 that are substrate-free and those that have bound ~ u b s t r a t e . l ~Greater *'~ differences have been observed between the CO binding constants of cytochromes P450 and model heme c o m p l e x e ~ . ' Several ~ ~ ~ ~ factors have been considered to contribute to the differences in ligand-binding properties of hemoproteins, including steric interactions between CO and the p r ~ t e i n , * axial ~-~~ ligand train,^^,^^ solvent effects,2s,26ligand stabilization through distal side effect^,^'-^* and electronic effects associated with the porphyrin (cis or the axial ligand (trans While extensive model heme studies h a v e considered the importance of these parameters in studies of oxygen-carrying heme proteins, comparatively few studies h a v e described t h e ligandbinding properties of model heme complexes for the cytochromes P450.In t h e present s t u d y w e have examined cis and trans electronic effects associated with varying basicities of 2,4-disubstituted porphyrins and thiolate ligands, respectively. T h e results provide a basis for assessing various factors contributing to t h e ligandbinding properties of c y t o c h r o m e s P450. Experimental Section Materials. Gold Label grade N,N-dimethylacetamide (DMA), l-butanethiol, and thiophenol were purchased from Aldrich. Dibenzo-18crown-6 ether, benzyl mercaptan, methyl thioglycolate and 4-fluorothiophenol were obtained from Parish Chemical. Meso-, spirographis(2-formyl-4-vinyl-), and 2,4-diacetyldeuteroheme free acids were obtained from Porphyrin Products as their chloride salts. Protohemin chloride was obtained from Sigma Chemical. 2,4-Dicyanodeuteroporphyrinwas obtained from Midcentury-Man-Win Chemicals and 2-acetyl-4-deutero-
*To whom correspondence should be addressed
0020-1669/86/1325-3734$01.50/0
porphyrin was obtained courtesy of Dr. Kevin Smith ( U . of California-Davis), and the iron was inserted into the porphyrins acSato, R.; Omura, T. Cytochrome Pdi0;Academic: New York, 1978. White, R. E.; Coon, M. J. Annu. Reo. Biochem. 1980, 49, 315-356. Debrunner, P. G.; Gunsalus, I. C.; Sligar, S. G.; Wagner, G. C. Met. Ions Biol. Sysr. 1978, 7 , 241-275. Ullrich, V. J . Mol. Catal. 1980, 7 , 159-167. Stern, J. 0.;Peisach, J. J . Biol. Chem. 1974, 249, 7495-7498. Chang, C. K.; Dolphin D. J. A m . Chem. Soc. 1975, 97, 5948-5950. Collman, J. P.; Sorrell, T. N. J . A m . Chem. SOC.1975, 97, 4133-4134. Champion, P. M.; Stallard, 8. R.; Wagner, G. C.; Gunsalus, 1. C. J . A m . Chem. Soc. 1982, 104, 5469-5472. Hahn, J. E.; Hodgson, K. 0.;Anderson, L. A,; Dawson, J. H. J . B i d . Chem. 1982, 257, 10934-10941. Tang, S. C.; Koch, S.;Papaefthymiou, G. C.; Foner, S.; Frankel, R. B.; Ibers, J. A,; Holm, R. H . J . A m . Chem. Soc. 1976, 98, 2414-2434. Collman, J. P.; Sorrell, T. N.; Hoffman, B. M. J . A m . Chem. Soc. 1975, 97, 913-914. Ruf, H. H.; Wende, P.; Ullrich, V . J . Inorg. Biochem. 1979, 1 1 , 189-204. Peterson, J. A,; Griffin, B. W. Arch. Biochem. Biophys. 1972, 151, 427-433. Debey, P.; Hui Bon Hoa, G.; Douzou, P. FEBS Lett. 1973,32, 227-230. Omura, T.; Sato, R. J . Biol. Chem. 1964, 239, 2370-2378. Omura T.; Sato, R.; Cooper, D. Y.; Rosenthal, 0.;Estabrook, R. W. FEBS Lett. 1965, 24, 1181-1189. Gray, R. D. J . B i d . Chem. 1982, 257, 1086-1094. Tuckey, R. C.; Kamin, H. J . Biol. Chem. 1983, 258, 4232-4237. Chang, C. K.; Dolphin, D. Proc. Natl. Acad. Sei. C'.S.A. 1976, 73, 3338-3342. Traylor, T. G.; Mincey, T. C.; Berzinis, A. P. J . A m . Chem. Soc. 1981, 103, 7084-7089. Collman, J. P.; Brauman, J. I.; Collins, T. J.; Iverson, B.; Sessler, J. L. J. A m . Chem. Soc. 1981, 103, 2450-2452. Rombere. R. W.: Kassner. R. J. Biochemistrv 1979. 18. 5387-5392. Geibel, i.;Canon, J.; Campbell, D.; Traylor,T. G. j . A : Chem. Soc. 1978, 100, 3575-3585. Perutz, M. F. Nature (London) 1970, 228, 726-734. Suslick, K. S.; Fox, M. M. J . A m . Chem. Soc. 1983,105, 3507-3510. C h a w K.; Traylor, T. G. Proc. Natl. Acad. Sei. U . S . A . 1975, 72. 1166Tii70. . Phillips, S. E. V.; Schoenborn, B. P. Narure (London) 1981, 292, 81-82. Shaaneti, B. Nature (London) 1982, 296, 683-684. Kitagawa, T.; Ondrias, M. R.; Rousseau, D. L.; Ikeda-Saito, M.; Yonetani, T. Nature (London) 1982, 298, 869-870. Maxwell, J. C.; Caughey, W. S. Biochemistry 1976, I S , 388-396. Satterlee, J. D.; Teintze, M.; Richards, J. H. Biochemistry 1978, 17, 1456-1462. Fuchsman, W. H.; Appleby, C. A. Biochemistry 1979,18, 1309-1321. Caughey, W. S.; Eberspaecher, E.; Fuchsman, W. H.; McCoy, S.; Alben, J. 0. Ann. N . Y . Acad. Sci. 1969, 153, 722-737. Abbott, E. H.; Rafson, P. A. J . A m . Chem. SOC.1974, 96, 7378-7379. Stanford, M. A.; Swartz, J. C.; Phillips, T. E.; Hoffman, B. M. J . Am. Chem. Soc. 1980, 102, 4492-4499. Falk, J. E.; Phillips, J. N.; Magnusson, E. A. Narure (London) 1966, 212, 1531-1533. Alben, J. 0.;Caughey, W. S. Biochemistry 1968, 7 , 175-183. Sono. M.; McCray, J. A.; Asakura, T. J . Biol. Chem. 1977, 252, 7475-7482
0 1986 A m e r i c a n C h e m i c a l Society
Heme and (Thio1ato)heme Complexes
Inorganic Chemistry, Vol. 25, No. 21, 1986 3735
Table I. Soret Absorption Maxima" of Ferrous Heme Thiolates, Ferrous Cytochromes P4s0,and Their C O Complexes syst
meso
Proto
heme thiolateb heme thiolate + C o b
397 372 450 402 358 434
41 1 383 464 408 364 446
P,,,-cam P,so-cam I?
nm.
+ CO
2-acetyl4-deutero
2-formyl4-vinyl
diacetyldeutero
dicyanodeutero
413 381 465
414 380 474
432 391 480 43 1 368 463
420 391 473
ref this work this work this work 56 56 56
In dimethylacetamide.
cording to the method of Chang et al.52 Carbon monoxide was obtained as Matheson Purity grade, and nitrogen as Linde oxygen-free grade. Matheson Model 8 and Model 19 (high purity) regulators used with C O and N2 cylinders were connected via stainless-steel tubing and fittings to hypodermic needles (Hamilton point style #3). Reagents were mixed in cuvettes made from 1 cm X 1 cm square glass tubing fitted to Ace #7663-06 glass sockets stoppered with two Alltech #6512 blue septa. All transfers were made via cannula or gas-tight syringes. Reduced Heme. A 10-pL sample of a concentrated sodium dithionite solution in 0.05 M phosphate buffer at pH 7.0 was added to a 2.5-mL preevacuated solution of heme in DMA in the glass cuvette described above. The resulting solution was subjected to three freeze-pumpthaw cycles after which nitrogen was added to atmospheric pressure. Similar spectrophotometric titration results were obtained with heme that had been reduced with sodium dithionite-crown ether complex53 in DMA, indicating that the trace amount of aqueous pH 7 buffer had no effect on the equilibria. Potassium Thiolate-Crown Ether Complex. Equimolar additions of KOH, thiol, and crown ether were mixed according to the method of Chang and D01phin.l~Reagents were mixed as follows: deaerated DMA was trensferred via cannula to a nitrogen-filled cuvette containing K O H pellets dissolved in a minimum amount of water (
