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Formation of a Square-Planar Co(L) B12 Intermediate Implications for Enzyme Catalysis

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Formation of a Square-Planar Co(L) B12 Intermediate Implications for Enzyme Catalysis Formation of a square-planar Co(l) B12 intermediate Implications for enzyme catalysis Michael D. Wirt, Irit Sagi, and Mark R. Chance Department of Chemistry, Georgetown University, Washington, DC 20057 USA ABSTRACT X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(l) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(l) and Co(ll) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co - N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co - N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co - C bond homolysis occurs, the Co - N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin- dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co - C bond cleavage) favors formation of the four-coordinate square-planar Co(l) species while inhibiting formation of the five-coordinate Co(ll) B12 complex. This paper presents the first direct evidence that formation of the Co(l) B12 intermediate must involve breaking of the Co -N (DMB) bond. INTRODUCTION 5'-Deoxyadenosylcobalamin (AdoCbl) (Fig. 1) functions nism of Co - C bond cleavage and the formation of as an essential cofactor for enzyme reactions involving four-coordinate Co(I) B12 intermediates. 1,2 exchange of a hydrogen atom and a second func- tional group, such as - NH2 in ethanolamine ammonia lyase or - OH in diol dehydrase (1). The mechanism of MATERIALS AND METHODS Co - C bond cleavage for these reactions is well estab- Materials lished as homolytic, forming a primary alkyl radical and the Co(II) B12 intermediate (2). In addition, valuable in- Cyanocobalamin and sodium borohydride were obtained from Sigma formation regarding the mechanism ofCo(II) B12 forma- Chemical Co. (St. Louis, MO). Aluminum oxide and 5,10,1 5,20-tetra- tion has been derived from bond dissociation energy phenyl-21H,23H-porphine Co(II) (CoTPP) were purchased from Al- drich Chemical Co. (Milwaukee, WI). Glycerol was obtained from studies ofHalpern and others (2-5). In these studies, the Fisher Scientific Co. (Pittsburgh, PA). All were used without further primary role of AdoCbl is identified to be that of a free purification. Cyanocobalamin purity was verified by optical spectros- radical precursor. Although nonhomolytic cleavage copy. We tested the glycerol, borohydride, and aluminum oxide in mechanisms for B12-dependent enzymes have been pro- solution and solid form for the presence of cobalt by x-ray absorption posed for years, only recently has substantial work been before sample preparation. No detectable edge jump was observed for the test samples; therefore, the cobalt concentration in these samples completed on two methylcobalamin (MeCbl)-depen- was <0.5% of that in the experimental samples and could be ignored. dent enzymes that are believed to proceed through a Co(I) B12 intermediate (6-8). Sample preparation We present structural data, derived from extended x- Co(I) B12 was prepared by reduction of 1.5 ml of 12 mM cyanocobala- ray absorption fine structure (EXAFS) and x-ray edge min with 0.7 ml of0.8 M NaBH4 under argon as described by Dolphin spectroscopy, that definitively demonstrate a four-coor- (9). The cyanocobalamin and NaBH4 solutions were degassed with dinate (distorted) square-planar configuration for Co(I) argon for 30 min before reduction. Co(II) B12 was formed from the B12. Forboth homolytic and heterolytic reactions, knowl- addition of 0.5 ml of NaBH4 to 1.5 ml of 12 mM cyanocobalamin. edge of the mechanism of Co - C cleavage and the Co(I) B12 was prepared via the Co(II) intermediate, by adding an addi- tional 0.2 ml of 0.8 M NaBH4 solution, under argon, to the yellow- structure ofthe intermediates is critical to understanding brown solution of Co(II) B12 with constant stirring. The Co(II) B12 how cleavage is accelerated by enzymes. A key question solution immediately turned to the dark blue-green Co(I) B12 species concerns the structural features ofalkyl cobalamins that (9). All samples were prepared as solutions in 35% glycerol to reduce are regulated by enzymes to favor homolysis versus het- sample cracking on freezing, with the exception ofCoTPP, which was a erolysis. Modulation ofthe Co - 5,6-dimethylbenzimid- solid. All samples were placed in 25 x 2.5 X 2-mm-deep lucite sample holders covered with mylar tape (approximate volume 200 ,l). To min- azole (DMB) bond may be a key factor in the mecha- imize fluorescence saturation effects, solid samples were diluted with aluminum oxide and ground to a fine powder in a mortar and pestle. The mixed powder was packed in a lucite sample holder. All solid Address correspondence to Mark R. Chance at the Department of samples were diluted 1:10 by weight in aluminum oxide. The liquid Physiology and Biophysics, Albert Einstein College of Medicine, samples were anaerobically transferred into the sample holders under Bronx, New York 10461. argon and immediately submerged in liquid nitrogen. 41.0639/20/1/6 oit 412 W06-3495/92/08/412/06 $2.00 Biophys.6J. Biophysical Society Volume63 August 1992 412-417 beam line X-9A, using a double flat Si(l I 1) crystal monochromator with fixed exit geometry. All experiments were carried out at 140-150 K, and sample temperature was maintained by flowing cooled nitrogen gas through a low-temperature cryostat as described previously (12). X-ray edge data having 3 eV resolution were recorded by counting at a specific energy for 2 s and incrementing the energy by 0.5 eV from 20 eV below the cobalt edge to 50 eV above the edge. EXAFS data, also having 3 eV resolution, were recorded by counting at a specific energy for 4 s and incrementing the energy by 10 eV from 120 eV below the cobalt edge to 20 eV below the edge, then in 1.0 eV steps to 15 eV above the edge, and finally in 3.0 eV steps from that point to 600 eV above the edge. A total of 15 scans were collected from three different samples. Photon flux was 3.75 X 10" photons/s at 100-ma beam current (13); both EXAFS and x-ray edge data were generally taken in the range of 90-180 ma. Cyanocobalamin was used as a standard to account for any shifts in the monochromator. K-a cobalt fluorescence was detected with a zinc sulfide-coated photomultiplier tube (14), and incident pho- ton scattering was rejected by an iron oxide filter. Output signals were amplified with a Keithley amplifier, converted to frequency, and counted in a scaler interfaced to a PDP 11/23+ Digital computer via CAMAC. For reference signals, mylar tape was mounted at a 450 angle to the x-ray beam to scatter photons counted by a similar photomulti- plier tube positioned perpendicular to the x-ray beam. This method provided excellent linearity between the sample and the reference de- tectors. Data analysis and errors FIGURE 1 Structure of AdoCbl. EXAFS background removal, k3-weighting, Fourier filtering, and non- linear least-squares fitting followed standard procedures (15-17). Raw, unsmoothed, undeglitched data were used for the analysis. Background X-ray absorption data were collected on several Co(I) B,2 samples subtracted data were Fourier transformed with use of cosine-squared that were characterized before and after data collection by optical spec- tapered windows as described previously (15). The taper ends had troscopy. Optical characterization was accomplished by transferring centers of 1.5 and 10.2 A-' in k-space. The Fourier filter back-trans- concentrated samples under argon to stoppered cuvettes containing form window taper centers were set to 0.8 and 1.9 A-'. At k, - Dk,/2 + deionized water that were previously degassed for 15 min. Immediately and k2 + Dk2/2, the window function tailed to zero, and at k, Dk,/2 after sample transfer, the cuvette was sealed, and an optical spectrum and k2 - DkJ2, the window function was completely square and left was taken. The Co(I) B,2 spectra were consistent with earlier absorption unchanged (where D is an arbitrary value that simply selects the degree spectra ofBeaven and Johnson (10). Percent conversion from cyanoco- oftaper). The back-transform (rwindow function) was the same type as the k window function. The taper functions were set at Dk, = Dk2 = 3.0 balamin to Co(I) B,2 was calculated at 93 ± 2%. The presence ofcyano- cobalamin in the Co(I) B,2 samples was measured by observing the A-'. The Fourier filter window taper functions were set to Dr, = Dr2 = remaining height of the 361-nm cyanocobalamin parent peak. This 0.2 A. All data were fit from 3.5 to 10.0 A' in k-space. The EXAFS was accomplished by fitting a cubic polynomial to the baseline of the solution was obtained by fitting the Co(I) B,2 data to CoTPP, which has pure cyanocobalamin and Co(I) B,2 spectra to normalize the peak a well-known crystal structure (18), by means of the University of heights. The absolute peak height was determined, allowing calculation Washington EXAFS package on the Georgetown 8700 VAX computer of the percent cyanocobalamin remaining in the Co(II) species (5% by (15).
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