Resonance Raman Spectra Show That Coenzyme B 12 Binding to Methylmalonyl-Coenzyme A Mutase Changes the Corrin Ring Conformation but Leaves the Co-C Bond Essentially Unaffected Shoulian Dong, ² Raghavakaimal Padmakumar, ‡ Nilesh Maiti, ‡ Ruma Banerjee,* ,‡ and Thomas G. Spiro* ,² Department of Chemistry, Princeton UniVersity Princeton, New Jersey 08544 Biochemistry Department, UniVersity of Nebraska Lincoln, Nebraska 68588 ReceiVed May 7, 1998 We report the first resonance Raman (RR) spectra of coenzyme B 12 in an active B 12 -dependent enzyme, methylmalonyl coenzyme A (MMCoA) mutase. The spectra reveal a large change in the corrin ring conformation, consistent with X-ray crystallography. 1 However, isotope labeling reveals the Co-C force constant to be only slightly altered, despite the dramatic activation of this bond in the catalytic cycle of this enzyme. 2 The Co-C bond is found in two enzyme cofactors, methyl- cobalamin (MeCbl) and 5′-deoxyadenosylcobablamin (AdoCbl). 3 The enzymes utilizing MeCbl catalyze methyl transfer reactions, 4 while those employing coenzyme B 12 (or AdoCbl, Figure 1) catalyze rearrangement reactions. 5 Among the latter, MMCoA mutase is the only isomerase found in both bacteria and mammals. It catalyzes the conversion of methylmalonyl coenzyme A to succinyl coenzyme A (Scheme 1). The rearrangement reaction is initiated by homolytic cleavage of the Co-C bond, forming an adenosyl radical and cob(II)- alamin. 5 The homolytic Co-C cleavage rate is dramatically en- hanced in B 12 -dependent enzymes. Upon substrate binding to the enzyme, the rate of appearance of Co(II) is 60 s -1 for diol dehydrase, 6 g300 s -1 in ethanolamine ammonia-lyase, 7 and g600 s -1 for MMCoA mutase, 8 compared to 4 × 10 -10 s -1 for AdoCbl in aqueous solution at 25 °C. 9 This ∼10 12(1 rate enhancement of Co-C bond homolytic cleavage amounts to an ∼15.5 kcal/mol destabilization of the Co-C bond. 9 However, the Co-C stretch- ing frequency for AdoCbl is nearly as high (424 cm -1 ) in MMCoA mutase as it is in aqueous solution (430 cm -1 ) (Figure 2). To minimize photolysis, the RR spectra were obtained on frozen samples by laser excitation at 568.2 nm, on the low energy side of the complex AdoCbl absorption spectrum (Figure 3). The absorption bands arise from multiple π-π* electronic transitions of the corrin ring, 10 and corrin vibrational modes dominate the RR spectra. 11,12 Nevertheless, the Co-C stretch (ν Co-C ) is enhanced, 13 albeit weakly, along with a deformation mode of the adenosyl ribose ring (δ ribose ) at 568 cm -1 , 14 which involves motion of the Co-bound C5′ atom (Figure 1). These bands are readily detected by 13 C5′ substitution (Figure 2). While the δ ribose mode is unaffected by enzyme binding, and ν Co-C is shifted only 6 cm -1 , some of the corrin RR bands display major changes in either frequency or intensity. These changes are evident in both the low-frequency (Figure 2) and high- frequency (Figure 4) regions, implying a change in the corrin conformation. We note that the RR changes are much larger than could be suggested by the relatively minor shape change of the absorption spectrum (Figure 3). Moreover the same RR changes are seen for the hydroxocobalamin product of photolysis (data not shown) although the absorption spectra (Figure 3, bottom) are again very similar for the free and enzyme-bound cofactor. Although quantitative interpretation will require a normal coor- dinate analysis, it is significant that all the modes above 1480 cm -1 shift up, by 3-11 cm -1 in the spectrum. We interpret this pattern as resulting from a flattening of the macrocycle, a trend that has been documented in metalloporphyrins. 15 The crystal structure of MMCoA mutase 1 does indeed reveal a flattening of the corrin relative to its strongly ruffled conformation in enzyme- free AdoCbl. 16 The highest frequency RR band, at ∼1600 cm -1 , is especially sensitive, and it probably arises from a mode involving out-of-phase stretching of adjoining meso-bridge bonds, similar to ν 10 or ν 19 in porphyrins, 15 which are also especially sensitive to conformation. For such a mode, the frequency upshift on flattening results from enhanced kinematic interaction of the adjoining bonds. Because of the steric crowding in AdoCbl, an enzyme induced change in the corrin conformation could weaken the Co-C bond via nonbonded forces. The effect of such forces is illustrated by the dramatically higher ν Co-C , 506 cm -1 , when the bulky adenosyl group is replaced by a methyl group, in MeCbl. 11,12 The force * Authors to whom correspondence should be addressed. ² Princeton University. ‡ University of Nebraska. (1) Mancia, F.; Keep, N. H.; Nakagawa, A.; Leadley, P. F.; McSweeney, S.; Rasmussen, B.; Bo ¨secke, P.; Diat, O.; Evans, P. R. Structure 1996, 4, 339-350. (2) Hay, B. P.; Finke, R. G. J. Am. Chem. Soc. 1987, 109, 8012-8018. (3) Banerjee, R. Chem. Biol. 1997, 4, 175-187. (4) Ludwig, M. L.; Matthews, R. G. Annu. ReV. Biochem. 1997, 66, 269- 313. (5) Halpern, J. Science 1985, 227, 869-875. (6) Valinsky, J. E.; Abeles, R. H.; Fee, J. A. J. Am. Chem. Soc. 1974, 96, 4709-4710. (7) Babior, B. M.; Moss, T. H.; Orme-Johnson, W. H.; Beinert, H. J. Biol. Chem. 1974, 249, 4537-4544. (8) Padmakumar, R.; Padmakumar, R.; Banerjee, R. Biochemistry 1997, 36, 3713-3718. (9) Hay, B. P.; Finke, R. G. J. Am. Chem. Soc. 1986, 108, 4820-4829. (10) Giannotti, C. In B12; Dolphin, D., Ed.; Wiley-Interscience: New York, 1982; Vol. 1, pp 393-430. (11) Dong, S.; Padmakumar, R.; Banerjee, R.; Spiro, T. G. J. Am. Chem. Soc. 1996, 118, 9182-9183. (12) Dong, S.; Padmakumar, R.; Banerjee, R.; Spiro, T. G. Inorg. Chim. Acta 1998, 270, 392-398. (13) The 440/430 cm -1 pair of peaks observed in the difference spectrum between natural abundance AdoCbl and 5′-CD2 AdoCbl in refs 11 and 12 are not observed with 5′- 13 C labeled AdoCbl. Instead, only the 430 cm -1 peak is observed. Therefore, the 440 cm -1 peak does not represent the νCo-C of an alternate conformer, but probably is due to a strong corrin ring mode coupled to 5′-CH2. (14) This mode is upshifted to 600 cm -1 in 2′,5′-dideoxyadenosylcobalamin in which the 2′-OH group is substituted by a hydrogen atom. Dong, S.; Padmakumar, R.; Banerjee, R.; Spiro, T. G. Munuscript in preparation. (15) Spiro, T. G.; Li, X.-Y. In Biological Applications of Raman Spec- troscopy; Spiro, T. G., Ed.; Wiley & Sons: New York, 1988; Vol. 3, pp 1-37. (16) Bouquiere, J. P.; Finney, J. L.; Lehmann, M. S.; Lindley, P. F.; Savage, H. F. J. Acta Crystallogr. 1993, B49, 79-89. Figure 1. Structure of coenzyme B12, AdoCbl. Scheme 1. Isomerization of MMCoA Catalyzed by MMCoA Mutase 9947 J. Am. Chem. Soc. 1998, 120, 9947-9948 S0002-7863(98)01584-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/12/1998