Electron paramagnetic resonance studies of cob(II)alamin and cob(II)inamides

Bayston, James H.; Looney, F.D.; Pilbrow, John R.; Winfield, M.E.

doi:10.1021/bi00812a020

Cited by 79 publications

(62 citation statements)

References 13 publications

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“…3C). These data are in agreement with published spectra of cob(II)inamides and cob(II)alamins, which like cob(II)yrinic acid, share the same UV-Vis and EPR spectra (27). The EPR spectrum of cob(II)yrinic acid seems base-on, because the superhyperfine coupling constant is 18 G. This result may be attributed to the Tris buffer or contaminating imidazole [20 G for cob(II)inamide in MeOH].…”

Section: Resultssupporting

confidence: 92%

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B ₁₂ )

Moore

Lawrence

Biedendieck

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B 12 , adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the d yz orbital, rather than the typical d z 2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.cobalt | NMR | precorrin | tetrapyrrole | metabolism

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Section: Resultssupporting

confidence: 92%

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B ₁₂ )

Moore

Lawrence

Biedendieck

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…12 The pattern of eight negative resonances centered at g ∥ = 2.0 and split by 144 G results from the unpaired electron localized in the Co 3d z2 -based orbital interacting with the spin of the 59 Co nucleus (I = 7 / 2 ). 62,63 The lack of any additional fine structure due to coupling from a 14 N nucleus (I = 1) confirms that the CFeSP-bound cofactor exists in the base-off conformation where the MBI ligand is dissociated, thus opening a potential coordination site for either a solvent molecule or a CFeSP-active site residue. Parallel experiments using Co 2+ CFeSP resuspended in H 2 17 O enriched buffer yielded an EPR spectrum suffering from significant broadening and an ~60% decrease in intensity throughout the g ∥ region (2800-4000 G) compared to the spectrum obtained in H 2 16 O (Figure 3).…”

Section: Epr Studies Of Co 2+ Cfespmentioning

confidence: 94%

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

et al. 2006

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Methyl transfer reactions are important in a number of biochemical pathways. An important class of methyltransferases uses the cobalt cofactor cobalamin, which receives a methyl group from an appropriate methyl donor protein to form an intermediate organometallic methyl-Co bond that subsequently is cleaved by a methyl acceptor. Control of the axial ligation state of cobalamin influences both the mode (i.e., homolytic vs heterolytic) and the rate of Co-C bond cleavage. Here we have studied the axial ligation of a corrinoid iron-sulfur protein (CFeSP) that plays a key role in energy generation and cell carbon synthesis by anaerobic microbes, such as methanogenic archaea and acetogenic bacteria. This protein accepts a methyl group from methyltetrahydrofolate forming Me-Co 3+ CFeSP that then donates a methyl cation (Me) from Me-Co 3+ CFeSP to a nickel site on acetyl-CoA synthase. To unambiguously establish the binding scheme of the corrinoid cofactor in the CFeSP, we have combined resonance Raman, magnetic circular dichroism, and EPR spectroscopic methods with computational chemistry. Our results clearly demonstrate that the MeCo 3+ and Co 2+ states of the CFeSP have an axial water ligand like the free MeCbi + and Co 2+ Cbi + cofactors; however, the Co-OH 2 bond length is lengthened by about 0.2 Å for the protein-bound cofactor. Elongation of the Co-OH 2 bond of the CFeSP-bound cofactor is proposed to make the cobalt center more "Co 1+ -like", a requirement to facilitate heterolytic Co-C bond cleavage.

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“…Furthermore, each cobalt hyperfine feature is associated with additional 14 N hyperfine features (I ϭ 1) apparent as triplets with 18 G coupling. The nitrogen hyperfine is associated with an axial ligand to cob-(II)alamin (24), either an imidazole from the DMB moiety of the cofactor (Fig. 1) or a histidine-derived protein ligand.…”

Section: Generation Of Cob(ii)alamin and Paramagnetic Species During mentioning

confidence: 99%

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii

Lawrence¹,

Gerfen²,

Sámano³

et al. 1999

Journal of Biological Chemistry

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The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5-triphosphates to 2-deoxynucleoside 5-triphosphates and uses coenzyme B 12 , adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2-deoxy-2-methylenecytidine 5-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.There are three classes of adenosylcobalamin (AdoCbl) 1 (Fig. 1, 1) requiring enzymes (1): those that catalyze reversible carbon skeleton rearrangements (class I), those that catalyze irreversible heteroatom elimination reactions (class II), and those that catalyze rearrangements involving migration of an amino group (class III). All three classes initiate their radicaldependent reactions by catalyzing carbon-cobalt bond homolysis. The mechanism(s) by which these enzymes catalyze C-Co homolysis of 1, the identification of the axial ligand trans to the C-Co bond that is cleaved, and its role in this process have recently been a topic of interest in many laboratories (2). The x-ray structures of methylmalonyl-CoA (MMCoA) mutase (3,4) and EPR studies of cob(II)alamin (2) bound to other class I enzymes (1, 5, 6) revealed that in each case the dimethylbenzimidazole (DMB) moiety of the cofactor is replaced by a protein-derived histidine. On the other hand, in diol dehydrase (DD), typical of the class II enzymes, DMB remains ligated to 2 (7,8). Unique among the AdoCbl-requiring enzymes is ribonucleotide reductase, the only enzyme that does not catalyze a rearrangement reaction and in which C-Co homolysis occurs concomitant with the formation of a protein-based thiyl radical (9 -11). The mechanism of homolysis and the identity of the axial ligand in this enzyme have not previously been addressed and are the subjects of this paper.Our laboratory has long been interested in the ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii. In the presence of a nucleoside 5Ј-triphosphate (NTP) substrate, a reducing system, and a 2Ј-deoxynucleoside 5Ј-triphosphate (dNTP) allosteric effector, RTPR catalyzes homolysis of the C-Co bond of 1 to generate 2, 5Ј-deoxyadenosine, and a thiyl radical at Cys-408 of RTPR in a kinetically competent and concerted fashion (9,10,(12)(13)(14). This thiyl radical is then proposed to initiate the nucleotide reduction chemistry by abstraction of the 3Ј-hydrogen atom of the NTP (15). In this paper we report studies with a mechanism-based inhibitor of this protein, 2Ј-deoxy-2Ј-methylenecytidine 5Ј-triphosphate (MdCTP, 3). These studies in conjunction with [U- EXPERIMENTAL PROCEDURESMaterials and Methods-Alkaline phosphatas...

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Electron paramagnetic resonance studies of cob(II)alamin and cob(II)inamides

Cited by 79 publications

References 13 publications

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B ₁₂ )

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B ₁₂ )

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii

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Electron paramagnetic resonance studies of cob(II)alamin and cob(II)inamides

Cited by 79 publications

References 13 publications

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B 12 )

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B 12 )

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii

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Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B ₁₂ )

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B ₁₂ )