Proc. Natl. Acad. Sci. USA Vol. 78, No. 5, pp. 2652-2656, May 1981 Chemistry

vi cation radicals of ferrous and free base isobacteriochlorins: Models for and (nitrite and sulfite reductases/enzymatic intermediates/macrocycle oxidations/electron spin resonance/molecular orbital calculations) C. K. CHANGa, L. K. HANSONb, P. F. RICHARDSONb, R. YOUNGa, AND J. FAJERbC bDepartment of Energy and Environment, Brookhaven National Laboratory, Upton, New York 11973; and aDepartment of Chemistry, Michigan State University, East Lansing, Michigan 48824 Communicated by Gerhart Friedlander, January 9, 1981

ABSTRACT Theoretical and experimental optical, redox, and C02H paramagnetic results are presented for models of siroheme, the COH iron isobacteriochlorin prosthetic group of nitrite and sulfite re- HO20 X H X~~~~~~HH ductases, and of sirohydrochlorin, the metal-free siroheme that H3C' N N _CO2H N. is an intermediate in the biosynthetic pathway to . The facile oxidation of many isobacteriochlorins, which distinguishes them from and , suggests that the siroheme HOC O2H macrocycle itself may undergo oxidation in the multi-electron en- CO2H CO2H zymatic cycles that reduce nitrite to ammonia and sulfite to hy- a b drogen sulfide. Extended Huckel MO calculations (i) help ration- alize the redox properties of isobacteriochlorins compared with FIG. 1. Structural formulas ofsiroheme (a) and 2,4-Me2Et8iBC (b). those of porphyrins and chlorins; (ii) indicate that Fe(IH) pyridine (See Fig. 4 for structures of the other isomers.) carbonyl [(py) (CO)] complexes ofisobacteriochlorins, unlike those of porphyrins and chlorins, should undergo oxidation from the macrocycle rather than the metal to yield i1 cation radicals; (iii) sented preliminary results suggesting that oxidation of some suggest that, in hexacoordinated Fe(II) isobacteriochlorin com- Fe(II)iBC complexes leads not to Fe(III) species but rather to plexes, the site of oxidation-i.e., the metal or the macrocycle- abstraction of an electron from the ir system to give Fe(II) ir will depend on the ligand field induced by the axial ligands; and cation radicals (24). (iv) predict similar unpaired spin density profiles for metal-free We describe here charge-iterative extended Huckel MO cal- and (py) (CO)Fe(H) isobacteriochlorin radicals. Experimental data culations that (i) provide a rationale for the redox properties of for three isomeric free-base and (py) (CO)Fe(II) complexes of di- iBCs compared with those ofporphyrins, chlorins, and bacter- methyloctaethylisobacteriochlorins support the theoretical calcu- iochlorins (BCs); (ii) indicate that ferrous pyridine-carbonyl lations and establish the existence of Fe(II) isobacteriochlorin 17 cations in vitro. [(py) (CO)Fe(II)] complexes ofiBCs, unlike those ofporphyrins or chlorins, should yield 7r cation radicals on oxidation; and (iii) Isobacteriochlorins (iBCs), porphyrins in which two adjacent predict that the unpaired spin density profiles ofmetal-free and rings are reduced, have recently elicited (1-9) consid- (py) (CO)Fe(II)-iBC radicals should be similar. Experimental erable interest because the prosthetic groups of sulfite and ni- redox, optical, and ESR data presented herein for three iso- trite reductases contain siroheme (10), an iron-iBC that has meric models ofsiroheme and sirohydrochlorin support the the- eight carboxylic acid side chains (Fig. la). The enzymes catalyze oretical calculations and unambiguously establish the existence (10) the six-electron reductions ofsulfite to hydrogen sulfide and of Fe(II)-iBC ir cations. nitrite to ammonia. In green plants, the latter reaction, N02 + 8H+ + 6e -- NH4+ + 2H20, is light driven with photo- METHODS synthetically reduced pyridine dinucleotide serving as electron The three isomeric iron dimethyloctaethyl iBCs [Fe(II)- donor to the active enzyme (11): Me2Et8iBCs]-2,8-dimethyl-2',8'-dihydro-3,3',7,7', 12,13, 17, 18-octaethylporphyrin [1,4-Fe(II)-Me2Et8iBC]; 3,8-dimeth- -+ NADPH -+ flavoprotein yl-3',8'-dihydro-2,2',7,7',12,13,17,18-octaethylporphyrin [2, -- ferredoxin -+ nitrite reductase. 4-Fe(II)-Me2Et8iBC] (see Fig. lb); and 3,7-dimethyl-3', 7'-dihydro-2,2',8,8', 12,13,17,18-octaethylporphyrin [2,3- An additional biological role has been attributed (12, 13) to Fe(II)-Me2Et8iBC]-and their free bases were synthesized as iBCs with the realization that sirohydrochlorins, demetallated described by Chang (5) and by Chang and Fajer (3). The cation sirohemes, are intermediates in the biosynthetic pathway ofthe radicals ofthe free bases were obtained by one-electron transfer , vitamin B12. to the radical of zinc (14) Zn- A salient feature of the iBC skeleton is its ease of oxidation Ph4PORtClO4O. The (py) (CO)Fe(II) radicals were generated and difficulty of reduction compared with those of porphyrins by oxidation with iodine (3). The techniques used for and chlorins (1-3, 8). This facile oxidation has led us to propose obtaining the ESR spectra have been described (15). (1-6) that the siroheme macrocycle itself may undergo redox reactions in the multi-electron enzymatic reductions ofthe sub- Abbreviations: py, pyridine; Ph4POR, tetraphenylporphine; iBC, iso- strates to ammonia and hydrogen sulfide, and we have pre- bacteriochlorin; Me2Et8iBC, dimethyloctaethylisobacteriochlorin; BC, bacteriochlorin; HOMO, highest occupied molecular orbital; LUMO, The publication costs ofthis article were defrayed in part by page charge lowest unoccupied molecular orbital; E112, polarographic half-wave payment. This article must therefore be hereby marked "advertise- potential. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. CTo whom reprint requests should be addressed. 2652 Downloaded by guest on September 29, 2021 Chemistry: Chang et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2653 -7fF

-8

g9 eg Or w z

-J

am -10 00r - Pa - pyrrole N p' pyrrole N Pa, pyrrole N -.-pyrrole-N- -Ha~~~~~~~~~~~~~~~yrlNyrlp&_|/lLT______+aup Olu.(T(7r) II 2(7r)_.-.. '2u(71 -*-2--(7T)- --- - * a2u(r) "a o(r)l$ Y- -fF- flY Y.. f Y- A ,fAd_X rVTf-,m. z.9V.0orrmmergy levelloual AiaVOMuagram.LurfarwthOe I\ HOMOs and LUMOs ofporphine, ', -121 Zn iBC, and BC complexes of Zn(II). Oxida- Nr n tion results in abstraction of an electron from an "a" (ir) orbital whereas reduction CHLORIN ISOBACTERIOCHLORIN BACTERIOCHLORIN adds an electron to the lowest ir* orbitals.

The self-consistent charge-iterative extended Huckel pro- ment (1, 2, 14, 24-26). In addition, as the ligand is saturated, gram and parameters have been presented by Gouterman and the energy required for oxidation should parallel the energy of coworkers (16, 17). Iterations were continued until the esti- the HOMOsf and decrease in the order: (hard) porphine mated (input) and calculated (output) charges for each atom >chlorin >iBCBC (easy), as is observed experimentally (Ta- agreed to within 0.015 electrons. Coordinates for the aromatic ble 1). Conversely, Fig. 2 suggests that iBC will be the hardest portions of the porphine, chlorin, iBC, and BC skeletons are to reduce and that the other three complexes shouldhave nearly given in ref. 18. The bond distances ofthe reduced pyrrole rings equal reduction potentials. This conclusion is also verified ex- in chlorin, iBC, and BC were set at 1.452 A for C,3-C,3(6) and perimentally for a series oftetraphenyl and octaethyl derivatives 1.500 A for CO.Ca,(6). In the Zn complexes, the Zn atom was (Table 1 and refs. 1-3 and 8). displaced 0.33 A from the macrocycle plane (6, 19, 20) with The energy differences between the HOMOs and LUMOs Zn-N(pyrrole) = 2.08 A. In the Fe complexes, the Fe was kept shown in Fig. 2 also provide an indication of the energy of the in plane, with Fe-N(pyrrole) = 2.01 A, Fe-N(saturated pyrrole) first absorption band ofeach complex. This difference increases = 2.03 A, Fe-N(pyridine) = 2.10 A (21), and Fe-CO = 1.77 in the order BC < iBC-chlorin

Table 1. Redox and optical characteristics ofporphyrin derivatives Half wave First-absorption potential, V band* Compound El/2(ox) * ElV(red)t AE±,V eV rm .2.0 Pr ZnPh4P .0.77 -1.35 (28) 2.02 .2.12 585 ZnPh4C 0.60 -1.33 (28) 1.93 2.03 610 ZnPh4iBC 0.28 (1) -1.73 (1) 2;01 2.06 602 (2) X 1.0 . ZnPhiBC 0.18 (26) -1.28 (29) 1.46 1.64 755 (26) H2Ph4P *0.95 (28) -1.08 (30) 2.03 1.92 647 (31) 0.0 H2Ph4C 0.88 -1.12 (28, 30) 2.00 1.91 .650 400 .500 600 700 800 H2iBC 0.57 (1) -1.52 (1) 2.09 1.91 650 H2Ph4BC 0.40 (26) -1.10 (28, 30) 1.50 1.68 740 (26) A, nm Numbers in parentheses indicate references. Ph4P, tetraphenyl por- FIG. 3. Opticalabsorptionspectraof(py)(CO)Fe(ID-2,3-Me2Et8iBC phine; Ph4C, tetraphenylchlorin. in CH2C,2 () and ofits ircation radical obtained by iodineoxidation * In CH2012 1In dimethylformamide or butyronitrile. t In,CH202 or benzene (H2Ph4P). be probed experimentally via the protons on the reduced rings whose interactions with the unpaired spin density at the a car- obtained if imidazole, a more likely biological ligand, is used bons (Pc) is defined by the McConnell equation: aHKpccos20, instead ofpyridine.) where aH is the observed hyperfine coupling constant in gauss, Cyclic voltametry shows that the compound undergoes a re- K is a constant (100), and 6 is the dihedraLangle between the versible one-electron oxidation at 0.13 V vs. the saturated cal- 2p, orbital of the a carbon and the plane defined by the a and omel electrode, whereas oxidations of the.corresponding por- ,( carbons and the proton (24). In chlorins (24), 6 ranges be- phyrin or chlorin complexes are irreversible (3). The optical tween 30° and 450; therefore, the calculations predict that the spectrum obtained on oxidation ofthe' iBC with a stoichiometric two (3 protons on the reduced rings should exhibit large hy- amount of iodine is shown in Fig. 3. The oxidized species ex- perfine splittings. The three isomers of H2Me2Et8iBC allow hibits an infrared CO absorption band at 2010 cm-' (to.be com- each a carbon ofthe reduced ring to be "sampled" experimen- pared with a CO stretch of1970 cm-' in the parent compound), tally. As shown in Fig. 6, the ESR data.forthe free-base radicals indicating that the axial ligand has not been.lost on oxidation. verify the profile predicted by the calculations: the 1,4 isomer The free radical character of the oxidized product is evident displays spectra consonant with two equivalent protons with aH from its ESR spectrum, which consists of three partially re- = 7.4 G; the 2,3 isomer shows two equivalent protons with solved lines centered at g = 2.003 (Fig. 4). This spectrum can smaller coupling constants, aH = 5.0 G; and the 2,4 isomer readily be assigned to a w cation of the iBC macrocycle on the shows two inequivalent protons with aH = 5.0 and 7.4 G. [The -basis ofmolecular orbitalcalculations and additional ESR results smaller splittings expectedifrom the nitrogens, meso protons, obtained from the 1,4 and 2,4 isomers of the Fe(II)iBCs, and and methyl and methylene groups are readily accommodated from their metal-free counterparts. within the spectral envelope. Some of these splittings are re- Unpaired spin densities calculated for r cation radicals of solved (1, 2) in the ESR spectrum ofZn-Ph4iBCt and lend ad- free-base iBC with an electron abstracted from the a,. orbital ;ditional support to the MO calculations.] are shown in Fig. 5. Notable.features ofthe calkulations are the Unpaired spin densities calculated for a (py) (CO)Fe(II)-iBC sizable spin densitiescomputed for the a carbons ofthe reduced ir cation predict that the ferrous radical should exhibit ESR rings. [Similar profiles are obtained from self-consistent field parameters similar to those ofthe free base (see Fig. 5). As seen Pariser-Parr-Pople calculations (1, 2). These spin densities can in Fig. 4, the ESRspectra ofthe three iron-containing isomers

FIG. 4. Second derivative ESR spectra of three isomeric models of siroheme-(py) (CO)Fe(IID-Me2Et8iBCI I- in CH2Cl2. (Top) Experimental spectra at 25TC. (Bottom) Simulations that assume two protons with the splitting constants shown. (1 G = 10-' T.) Downloaded by guest on September 29, 2021 Chemistry: Chang et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2655

0 .002 mentally. (Note that the calculations predict a ir cation of the .009 009 C .002 .008-008 BC complex as well.) .010.014 .000 .000 .014 .011 The calculated energies of the HOMOs in the three com- .018 .067 R .045 .0011 .0 plexes also follow the observed oxidation potentials of 0.52, .070020201 .00.073 N~Fe- .067N 4.073 ~ .082) N N +.082 0.32, and 0.13 V for porphine, chlorin, and iBC, respectively .061 I8,/ .06 HH8 02.060 .0N N 57, .06 (3). (The first two are irreversible because the Fe(III) N N .04 .058 .058 .060 .060 do not bind CO: .034 .034 .034 complexes /.028 ,.000 \.036 .000 .036t (Noo (py)(CO)Fe(II)-porphine * (py)Fe(III)-porphine' + CO. +e FIG. 5. Unpaired spin densities calculated for (py)(CO)Fe(II) (Left) The top filled dorbital is approximately 0. I eV higher for chlorin and metal-free (Right) iBC ircations. The free-base values are the av- than for porphine. On further saturation ofthe macrocycle, the erage ofcomputations for two tautomeric forms. (The crystal structure alu orbital surfaces above the Fe orbitals in iBC (and BC), of 2,3-H2Me2Et8iBC shows the two inner protons to be.shared among thereby lowering the oxidation potential of the iBC complex. the four pyrrole nitrogens; unpublished results.) The experimental results thus clearly establish the existence parallel those found for the metal-free species and leave little of r cation radicals of iBCs, and the MO calculations provide doubt that electron abstraction has occurred from the macro- a rationale for both the redox properties of the complexes and cycle and not the metal. the sites ofoxidation. The energy level diagrams of Fig. 7 offer an explanation for Whether these results can be extrapolated to the enzymatic the very different modes ofoxidation ofporphine, chlorin, and cycles of nitrite and sulfite reductases will depend crucially on iBC iron complexes. The migration ofthe irorbitals as afunction the axial ligands imposed by the protein on the sirohemes. For of ring saturation parallels that of the Zn complexes. (Again, ligands that do not have lone pair or ir, r"* orbitals capable of changes in the charge density distribution between metal, ma- mixing strongly with and perturbing the metal .d orbitals, the crocycle, and axial ligands are minimal across the series.) energies of the latter will depend on two factors: (i) the ligand Whether an electron is abstracted from the metal or the ma- field strength ofthe ligands and (ii) the withdrawal or donation crocycle on oxidation of a low spin Fe(II) complex will depend ofelectrons to the iron. Increasing-the ligand field strength en- on the relative energies of the d ta and the highest filled por- larges the energy gap between the filled t2g and the empty eg ir metal orbitals, without altering the center of gravity, so that the t2g phyrin irorbitals. Ifa t2g orbital lies above the orbitals, orbitals move to lower energies and the eg orbitals move to oxidation will occur; if tc

FIG. 6. Second derivative ESR spectra ofthree isomeric models ofsirohydrochlorin-H2Me2Et8iBCt C104- in CH2Cl2. (Top) Experimental spec- tra at25°C. (Bottom) Simulations that demonstrate that the spectral patterns are determined by two protons with the splitting constants shown.(For optical spectra ofthe radicals, see refs. 1, 2, andS5.) g = 2.0025. Downloaded by guest on September 29, 2021 26562CProc.Chemistry: Chang et al. Natl. Acad. Sci. USA 78 (1981)

-7 -dz2 -dz2 -dz2

- Co 7*) Co(7r*) --Co orp in 7r* eddI f - d,2 2 d xy -8 y - x -y \pyridine (vr*) \pyr, r* , -- 7r*'

--"e9"-,y)-71=-( -_

-= eg (7T* *-- w z w -J FIG. 7. Energy level diagrams for o -10- pyridine-CO complexes of Fe(II)- 0 porphine, chlorin, iBC, and BC. Note that in the latter two compounds, the _H" /dX2_ y2 ali orbitals are the -highest occupied the Fe Ot"a (-W))------; while, in porphine and chlorin, - _ if orbitals lie highest. Oxidation will _,-, dyetdxz therefore result in Fe(II)-porphine or IU -- chlorin but in Fe(][) ir cations for iBC *'-, Y. y x and BC. The x and y axes are defined by the methine carbons for porphine -12L FeR and iBC and by the, pyrrole nitrogens for chlorin and BC. Thus, the ta, orbit- als are d,, dizz andd2-y2 for the former BACTERIOCHLORIN and dug, dy,.and 4Y for the latter.

phine, chlorin, and iBC are nearly identical (3) and, in contrast 12. Scott, A. I. (1978) Acc. Chem. Res. 11, 29-36. & to the (py)(CO) results, the ligand field gap calculated for 13. Battersby, A. R., McDonald, E., Thompson, M. Bykhovsky, V. Y. (1978) J. Chem. Soc. Chem. Commun., 150-151. bis(py)Fe(II)-iBC is considerably smaller with the t2g orbitals 14. Fajer, J., Borg, D. C., Forman, A., Dolphin, D. & Felton, R. H. pair placed at higher energies (unpublished results). Any ligand (1970)J. Am. Chem. Soc. 92, 3451-3459. with a ligand field strength comparable with that of (py)(CO) 15. Borg, D. C., Forman, A. & Fajer, J. (1976) J. Am. Chem. Soc. would therefore be expected to yield a ir cation on oxidation of 98, 6889-6893. the Fe(II)-iBC complex.' 16. Schaffer, A. M., Gouterman, M. & Davidson, E. R. (1973) Theor. Chim. Acta 30, 9-30. We thank Dr. A. Forman for the simulations ofFig. 4. This work was 17. Gouterman, M. (1978) in The Porphyrins, ed. Dolphin, D. (Ac- supported by the Division of Chemical Sciences, U.S. Department of ademic, New York), Vol. 3, pp. 1-166. Energy, under Contract DE-AC02-76CH00016 at Brookhaven National 18. Eaton, W. A., Hanson, L. K., Stephens, P. J., Sutherland, J. C. Laboratory and by National Science Foundation Grant CHE 7815285 & Dunn, J. B. R. (1978) J. Am. Chem. Soc. 100, 4991-5003. and U.S. Department ofAgriculture Grant 59-2261-0-1437-0 at Mich- 19. Spaulding, L. D., Andrews, L. C. & Williams, G. J. B. (1977)J. igan State University. Am. Chem. Soc. 99, 6918-6923. 20. Collins, D. M. & Hoard, J. L. (1970) J. Am. Chem. Soc. 92, 3761-3771. Similar effects in the isoelectronic osmium(II) porphyrins are dis- 21. Peng, S. M. & Ibers, J. A. (1976) J. Am. Chem. Soc. 98, cussed in ref. 33. 8032-8036. 1. Richardson, P. F., Chang, C. K., Spaulding, L. D. & Fajer, J. 22. Weiss, C. (1978) in The Porphyrins, ed. Dolphin, D. (Academic, (1979) J. Am. Chem. Soc. 101, 7736-7738. New York), Vol. 3, pp. 211-223. 2. Richardson, P. F., Chang, C. K., Hanson, L. K., Spaulding, L. 23. Maggiora, G. M. & Weimann, L. J. (1974) Int. J. Quant. Chem. D. & Fajer, J. (1979) J. Phys. Chem. 83, 3420-3424. Quant. Biol. Symp. 1, 179-195. 3. Chang, C. K. & Fajer, J. (1980)J. Am. Chem. Soc. 102, 848-851. 24. Fajer, J. & Davis, M. S. (1979) in The Porphyrins, ed. Dolphin, 4. Hanson, L. K., Chang, C. K., Davis, M. S. & Fajer, J. (1980) in D. (Academic, New York), Vol. 4, pp. 197-256. Interaction Between Iron and Proteins in Oxygen and Electron 25. Fajer, J., Davis, M. S., Brune, D. C., Spaulding, L. D., Borg, Transport, ed. Ho, C. (Elsevier/North-Holland, New York), in D. C. & Forman, A. (1976) Brookhaven Symp. Biol. 28, 74-104. press. 26. Fajer, J., Borg, D. C., Forman, A., Felton, R. H., Dolphin, D. 5. Chang, C. K. (1980) Biochemistry 19, 1971-1976. & Vegh, L. (1974) Proc. Nati. Acad. Sci. USA 71, 994-998. 6. Barkigia, K. M., Fajer, J., Spaulding, L. D. & Williams, G. J. B. 27. Fuhrhop, J.-H., Kadish, K. M. & Davis, D. G. (1973) J. Am. (1981) J. Am. Chem. Soc. 103, 176-181. Chem. Soc 95, 5140-5147. 7. Stolzenberg, A. M., Spreer, L. 0. & Holm, R. H. (1979) J. 28. Felton, R. H. (1978) in The Porphyrins, ed. Dolphin, D. (Aca- Chem. Soc. Chem. Commun., 1077-1078. demic, New York), Vol. 5, pp. 53-125. 8. Stolzenberg, A. M., Spreer, L. 0. & Holm, R. H. (1980)J. Am. 29. Oester, M. (1971) Dissertation (Univ. Wisconsin, Madison, WI). Chem. Soc. 102, 364-370. 30. Wilson, G. S. & Neri, B. P. (1973) Ann. N.Y. Acad. Sci. 206, 9. Montforts, F. P., Ofner, S., Rasetti, V., Eschenmoser, A., Wog- 568-578. gon, W. D., Jones, K. & Battersby, A. R. (1979) Angew. Chem. 31. Smith, K. M., ed. (1975) Porphyrins and Metalloporphyrins (El- Int. Ed. Engl. 18, 675-677. sevier, Amsterdam), pp. 871-889. 10. Murphy, M. J., Siegel, L. M., Tove, S. R. & Kamin, H. (1974) 32. Antipas, A., Buchler, J. W., Gouterman, M. & Smith, P. D. Proc. Nati. Acad. Sci. USA 71, 612-616. (1978) J. Am. Chem. Soc. 100, 3015-3024. 11. Vega, J. M. & Garrett, R. N. (1975) J. Biol. Chem. 250, 33. Antipas, A., Buchler, J. W., Gouterman, M. & Smith, P. D. 7980-7989. (1980) J. Am. Chem. Soc. 102, 198-207. Downloaded by guest on September 29, 2021