J . Chem. SOC., Faraday Trans. 1, 1982,78, 3393-3396 Formation of Manganese@) Porphyrin Derivatives from Manganese(II1) Derivatives by Ionizing Radiation BY RAMAKRISHNA RAO AND MARTYN C. R. SYMONS* Department of Chemistry, The University, Leicester LE 1 7RH AND ANTHONY HARRIMAN Davy Faraday Research Laboratory, The Royal Institution, London W 1X 4BS Received 26th March, 1982 Exposure of manganese(Ir1) porphyrin solutions to 6oCo prays at 77 K gave the manganese(I1) derivatives, with gI (apparent) z 6, A(55Mn) = 77 G and ID( 2 0.3 cm-I. The spectra are very similar to those for Mn" derivatives prepared chemically. Weak features in the g = 2 region for these complexes were obscured by features from a symmetrical Mn" derivative of unknown origin. These features grew in intensity on melting and re-freezing.These results suggest that low-temperature irradiation coupled with e.s.r. spectroscopy may be a suitable technique for detecting Mn"' derivatives in biological systems. Although manganese occurs quite widely in biological systems, many centres give no e.s.r. signals in the pure state. An important example is that of chloroplasts. We have established that the use of ionizing radiation at low temperatures (usually 77 K) is a powerful method for inducing specific electron addition,' and that this technique can usefully be applied to biological systems.2-6 It seemed possible that biological systems containing Mn1I1 ions, and therefore giving no detectable e.s.r. signals, might be conveniently converted into MnlI derivatives by simple electron addition under low-temperature conditions that should inhibit further reactions. The aim of this study was to test this hypothesis using MnlIr porphyrins as good model systems.The results show that the method is indeed feasible. Previous work7 has shown that MnlI1 porphyrins undergo very inefficient photoreduction in outgassed solution to form the corresponding MnIl porphyrin. Similar reductions can be achieved with chemical reductants,s such as dithionite, although both techniques have little application for biological samples. EXPERIMENTAL PREPARATION MnTSPP was prepared as described by Harriman and Porterg and purified by repeated chromatography on Dowex 50-wX8 cation-exchange resin followed by exhaustive dialysis against deionized water. MnTPP was prepared from tetraphenylporphyrin (chlorine-free, Aldrich) (1 g), dissolved in glacial acetic acid (80 cm3) and Mn(OAc), (4 g) was added.The mixture was refluxed for 4 h and then evaporated to dryness. The solid product was washed with water and dried overnight at 90 OC under vacuum. It was purified by repeated chromatography on alumina using CHCl, as eluent. 33933394 FORMATION OF MnI1 PORPHYRINS Dilute solutions (10-50 mmol dm-3) in methanol (CD,OD was used to minimise e.s.r. absorption from the solvent) were degassed and frozen as small beads in liquid nitrogen. They were exposed to 6oCo y-rays at 77 K in a Vickrad source to doses of 0.5-3.0 Mrad.* The e.s.r. signals assigned to Mn" grew steadily during this period. c 1000 G (9.0977 GHzI MI (55Mn) -"Z I I - 3/2 + 3200 G (9.0957 GHzI 50 G W H A I I I I I I MI ( Mn) -72 -% -% *% *3/2 +% FIG.1.-First derivative X-band e.s.r. spectra for a solution of Mn"' TSPP in CD,OD after exposure to 6oCo y-rays at 77 K. (a) At 77 K, showing the g = 6 feature assigned to species A (MnIITSPP) and (6) after annealing to the glass-point and re-cooling, showing features assigned to species B. (The main hyperfine features are indicated with MI values; the intermediate lines are forbidden transitions and the central feature is due to organic radicals.) E.s.r. spectra were measured on a Varian E-109 X-band spectrometer calibrated with a Hewlett-Packard 52461, frequency counter and a Bruker BH 12 E field probe standardised with a sample of diphenylpicrylhydrazyl. Samples were annealed by decanting the liquid nitrogen from the insert Dewar flask and monitoring the spectra continuously. Samples were re-cooled whenever significant spectral changes were observed.RESULTS AND DISCUSSION The solutions showed no e.s.r. features at 77 K before irradiation. After exposure, in addition to intense signals in the g = 2 region from *CD, and *CD,OD radicals, a broad resonance appeared in the g = 6 region which was just resolved into six hyperfine components from coupling to 55Mn nuclei ( I = $) [species A, fig. 1 (a)]. * 1 rad = J kg-I.R. RAO, M. C. R. SYMONS A N D A. H A R R I M A N 3395 After annealing to remove solvent features, two sets of lines from 55Mn in the g = 2 region were detected. One set, characteristic of Mn’I with only a small zero-field splitting, grew in intensity on annealing [species B, fig.l(b)]. At the same time, the other set, assigned to species A, decayed slightly and became less well resolved. TABLE 1 .- E.s.R. PARAMETERS FOR SOME MnI1 PORPHYRINS apparent values of ga 55Mn hyperfine couplingb complex g /I g, A / / A , Mn(II)TSPPC ca. 2.00 5.9 & 0.1 ca. 78 78-L- 1 Mn(I I)(TPP)(PY Id 2.00 5.96 74 74 a These are not true values of g (see text). G = lop4 T. D > 0.3 cm-’. D = 1.2 cm-1.8 SPECIES A Because of the great width of the e.s.r. features, the data given in table 1 have large error limits. Nevertheless, comparison of these results with results reported elsewhere for similar complexes7v8 show that simple electron addition must have occurred to give the MnII TSPP derivative.These results are typical of high-spin (S = 5) d5 systems with large tetragonal zero-field splitting parameters (D). The results require that D be greater than the microwave energy (ca. 0.3 ~ m - l ) . ~ Because of the absorption from species B, it was difficult to obtain details of the expected ‘parallel’ feature in the g = 2 region, but extra features, giving “gI1 ’’ z 2.00 and A,, z 78 G could be picked out. The g = 2 features are usually far weaker than the g = 6 features for such complexes and the hyperfine splitting is usually isotropic. We stress that the quoted values of g , , and gl are nothing to do with the true g-tensor components, but simply serve to identify transitions within the S = f manifold that are intense at X-band frequenciesg In fact, the values of g must be nearly isotropic at g = 2.00. We conclude that simple electron addition occurred on irradiation.The broadening observed on annealing presumably reflects some change in the environment as the Mnl* system relaxed to its equilibrium dimensions. It may be significant that the MnlI ion resides some 0.56 A above the plane of the porphyrin ring whereas the MnlI1 ion is much nearer to being in-the-plane. Consequently, upon reduction there must be a large geometry change and this may contribute towards the annealing effect. SPECIES B The e.s.r. spectrum for this species is typical of high-spin (S = g) MnI1 complexes with near cubic symmetry. In addition to the six main hyperfine components, intermediate lines due to formally forbidden transitions are apparent.The broadening of the hyperfine lines on going from low to high field indicates a small D component. Although this species is clearly a product of irradiation, it seems to us unlikely that it is formed directly from MnlI1 TSPP, since this would require the ejection of manganese from the porphyrin ring and resolvation. This is extremely unlikely in dilute solutions for which the only expected process is electron additi0n.l It is more likely that some MnlI1 impurity with a higher electron affinity than that of MnlI1 TSPP is involved, in which case species B is of no special significance.3396 FORMATION OF MnII PORPHYRINS CONCLUSION These results clearly establish that MnlI1 complexes which do not involve manganese clusters can be readily reduced by ionizing radiation, and the resulting MnIL complexes can be detected by e.s.r.spectroscopy in low concentration. Preliminary results with chloroplasts have not been successful. This may be because manganese pairs are present, as suggested by the recent work of Dismukes and Siderer.lo The success of the method with simple Mn porphyrins together with the failure to detect anything with chloroplasts suggests that more model systems are required. It should be possible to replace the haem in a natural protein, such as myoglobin, with a Mn porphyrin and so obtain a system intermediate between the simple porphyrins and the chloroplast. We thank the S.E.R.C. for a grant to D.N.R.R. M. C. R. Symons, Pure Appl. Chem., 1981, 53, 223. M. C. R. Symons and R. L. Petersen, Biochim. Biophys. Acta, 1978, 535, 241. M. C. R. Symons and R. L. Petersen, Biochim. Biophys. Acta, 1978,537, 70. M. C. R. Symons and R. L. Petersen, Biochim. Biophys. Acta, 1978, 535, 247. M. C. R. Symons and R. L. Petersen, J . Chem. Res. (S), 1978, 382; (M), 1978, 4572. 'I A. Harriman and G. Porter, J. Chem. Soc., Faraday Trans. 2, 1979, 75, 1543. I. A. Duncan, A. Harriman and G. Porter, J. Chem. Soc., Faraday Trans. 2, 1980, 76, 1415. A. Harriman and G. Porter, J. Chem. SOC., Faraday Trans. 2, 1979, 75, 1532. * M. C. R. Symons and R. L. Petersen, Proc. R. SOC. London, Ser. B, 1978, 201, 285. lo G. C. Dismukes and Y. Siderer, FEBS Lett., 1980,121,78; Proc. Natl Acad. Sci. USA, 1981, 78, 274. (PAPER 2/524)