The naturally occurring peptidyl protease inhibitor leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) has been prepared labeled with 13C at the argininal carbonyl. 13C chemical shift data for the trypsin-leupeptin inhibitor complex in the pH range 3.0-7.6 reveal the presence of two pH-dependent covalent complexes, suggestive of two interconverting diastereomers at the new asymmetric tetrahedral center created by covalent addition of Ser195 to either side of the 13C-enriched aldehyde of the inhibitor. At pH 7 two signals are observable at delta 98.8 and delta 97.2 (84:16 ratio), while at pH 3.0 the latter signal predominates. In the selective proton 13C-edited NOE spectrum of the major diastereomer at pH 7.4, a strong NOE is observed between the hemiacetal proton of the inhibitor and the C2 proton of His57 of the enzyme, thus defining the stereochemistry of the high pH complex to the S configuration in which the hemiacetal oxygen resides in the oxyanion hole. pH titration studies further indicate that the 13C chemical shift of the S diastereomer follows a titration curve with a pKa of 4.69, the magnitude of which is consistent with direct titration of the hemiacetal oxygen. Similar pH-dependent chemical shifts were obtained by using CPMAS 13C NMR, providing evidence for the existence of the same diastereomeric equilibrium in the solid state.

The structure of a novel tetradehydrocorrin, factor IV, isolated from Propionibacterium shermanii has been established by multidimensional NMR spectroscopy. Incorporation of radiolabeled factor IV into cobyrinic acid established the biointermediacy of this cobalt complex, whose structure has implications for the mechanisms of the anaerobic pathway to B 12 .In the aerobic bacterium Pseudomonas denitrificans, the biosynthetic pathway to vitamin B 12 , featuring an oxygenrequiring step, has been established (for reviews, see refs. 1 and 2), but it is now clear that a second, anaerobic route to corrin has existed in nature for ca. 4 billion years (2, 3). Thus, the obligate anaerobes, such as methanogenic bacteria, and the semianaerobic Propionibacterium shermanii are able to synthesize B 12 by processes that differ at several pivotal steps from the aerobic pathway. The Ps. denitrificans route uses a twostage ring contraction sequence on the metal-free substrate precorrin-3, using O 2 (4) to fashion ␥-lactone and tertiary hydroxyl functions at C-1 and C-20 in precorrin-3x (Scheme I) as a prelude to a pinacol-like rearrangement, whereas cobalt is inserted late, i.e., not until all S-adenosylmethionine (AdoMet)-derived methyl groups are in place and amidation of the carboxylates has begun (1, 2).In contrast, it has been found (5) that in Pr. shermanii cobalt is inserted early into precorrin-3, which is ring-contracted without O 2 as cofactor (Scheme I), and at a later, unknown stage, a unique exchange of carbonyl oxygen at the C-27 (ring A) acetate occurs (6, 7); this is another event that is not paralleled in aerobic metabolism (8). In this report, we describe the isolation of a new B 12 intermediate from Pr. shermanii whose surprising structure provides circumstantial but compelling evidence for those mechanisms operating during anaerobic biosynthesis that mediate ring contraction and loss of the 2-carbon fragment from the western side. MATERIALS AND METHODSPreparation of Factor IV from epi-Factor II. A cell-free extract of Pr. shermanii (from 50 g of wet cells) was prepared by sonication (50 min at 0-4ЊC) in phosphate buffer (pH 7.6; 50 ml). Glutathione (5 mg), ATP (22 mg), NAD (5 mg), NADH (10 mg), AdoMet (75 mol), EDTA (3.5 mol), Co 2ϩ (4 mol), and Co 3ϩ (1.8 mol) were added to the supernatant of the centrifuged suspension (17,000 ϫ g; 25 min at OЊC). For preparative isolation, 3-epi; 8-epi factor II ( Fig. 1; 500 mol) was added, and the incubation was carried out at 31ЊC for 16 hr. Sodium cyanide (5 mg) was then added, and the tetrapyrrolic mixture was adsorbed on DEAE-Sephadex A-25 (Pharmacia) followed by extraction and esterification by MeOH͞

The enzyme CbiL from the facultative anaerobe Salmonella typhimurium exhibits a high degree of homology to CobI from the aerobe Pseudomonas denitrificans (29% identity; 51% conservation obtained by a Blastp search of the ncbi database). As CobI catalyzes the third methylation in the aerobic pathway to vitamin B12 it is proposed that CbiL catalyzes the analogous step in the anaerobic pathway. Potential metallo and metal-free substrates were characterized and their redox states defined by a combination of physicochemical techniques (MALDI-MS, NMR, UV/vis, IR, and EPR) and then used to investigate the function of CbiL. CbiL exhibited an absolute requirement for the presence of a metal ion (Co(II), Ni(II), or Zn(II)) within the tetrapyrrole substrate. CbiL had no preference for the redox state of its cobalt tetrapyrrole substrate, methylating both the reduced form, Co(II) 2, 7-dimethyl-dipyrrocorphin (Co(II)-precorrin-2), and the oxidized form, Co(III) 2,7-dimethyl-isobacterioclorin (Co(III)-factor-II). In contrast CbiL had a marked preference for the oxidized Ni(II) and Zn(II)-2,7-dimethyl-isobacteriochlorin (Ni(II) and Zn(II)-factor-II). Removal of the metal ion from a product of CbiL (Zn(II)-factor-III) allowed characterization by 13C NMR, identifying the tetrapyrrole as 2,7,20-trimethyl-isobacteriochlorin (factor-3), indicating that CbiL methylates at C20, the same site as that methylated by CobI. Competition experiments, utilizing isotopic labeling to distinguish otherwise identical mass substrates and products, revealed that oxidized Co(III) or Ni(II)-factor-II were equally good substrates, whereas Co(II)-precorrin-2 was much preferred over Ni(II)-precorrin-2. Excess Ni(II)-precorrin-2 did not decrease CbiL methylation of Co(II)-precorrin-2, implying that CbiL has a low affinity for Ni(II)-precorrin-2. These results are interpreted on the basis of tetrapyrrole ruffling occurring on the optimization of the metallo-N bond distances. The greater flexibility of the reduced precorrin-2 ring system allows greater deformation on accommodating the bound metal ion, the distortions imposed by bound Ni(II) or Zn(II) ions being larger than Co(II). The resulting distortions imposed on the precorrin ring could then decrease catalysis by causing a departure from the optimal substrate conformation required for CbiL. On oxidation of the Ni(II) or Zn(II)-precorrin-2, the increased stiffness of the ring could then constrain the metallo-factor-II conformation toward that of the usual substrate, allowing greater methylation by CbiL. In contrast to its counterpart CobI in the aerobic pathway of B12 biosynthesis, which methylates the metal-free precorrin-2, these studies show CbiL to be the first methylase unique to the anaerobic pathway, methylating a metallo-precorrin-2 substrate. Implications of CbiL specificity for the mechanism of the anaerobic B12 pathway are discussed.