Spectroscopic studies of the corrinoid/iron-sulfur protein from Moorella thermoacetica - PubMed (original) (raw)

Spectroscopic studies of the corrinoid/iron-sulfur protein from Moorella thermoacetica

Troy A Stich et al. J Am Chem Soc. 2006.

Abstract

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 Me-Co3+ and Co2+ states of the CFeSP have an axial water ligand like the free MeCbi+ and Co(2+)Cbi+ cofactors; however, the Co-OH2 bond length is lengthened by about 0.2 angstroms for the protein-bound cofactor. Elongation of the Co-OH2 bond of the CFeSP-bound cofactor is proposed to make the cobalt center more "Co1+-like", a requirement to facilitate heterolytic Co-C bond cleavage.

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Figures

Scheme 1

Adapted from ref .

Figure 1

Chemical structure and numbering scheme for the CFeSP cofactor, Factor IIIm (methyl-Co3+–5′methoxybenzimidazolyl cobamide).

Figure 2

Comparison of 4.5 K Abs (top), CD (middle), and 7 T MCD (bottom) spectra of as-isolated Co2+CFeSP (solid lines) and Co2+Cbi+ (dotted lines).

Figure 3

Experimental (thick black lines) and simulated (thin red lines) X-band EPR spectra of Co2+CFeSP resuspended in natural abundance H2O (top) and H2 O (bottom). Inset: Exploded view reveals additional features in the high-field region of the EPR spectrum of Co2+CFeSP resuspended in H2 O (solid line) compared to that resuspended in n.a. H2O (dotted line). EPR spectra were recorded at 100 K using 9.39 GHz microwave frequency, 16 mW microwave power, 10.4 G modulation amplitude, and 100 kHz modulation frequency. Relevant EPR parameters and complete simulation input files are given in Table 2 and the Supporting Information, respectively.

Figure 4

Comparison of 4.5 K Abs (top), 4.5 K CD (middle), and 7 T 100 K MCD (bottom) spectra of methylated CFeSP (solid lines) and AdoCbi+ (dotted lines)

Figure 5

Low-frequency (top panel) and high-frequency (bottom panel) regions of the resonance Raman spectra obtained for MeCbl at pH 1.0 (A), 12CH3–CFeSP (B), 13CH3–CFeSP (C), and CD3–CFeSP (D). All data were collected at 77 K with 441.6 nm laser excitation

Figure 6

Calculated potential energy surfaces (A), Co–CH3 bond length (B), and nature of axial ligand bonding (C) as a function of Co–OH2 bond length for a series of QM/MM optimized MeCbi+ models.

Figure 7

Calculated MO diagram detailing the relative energies and compositions of the HOMO, LUMO, and orbitals involved in the dominant axial ligand bonding interactions for selected QM/MM optimized MeCbi+ models with lower axial ligand Co–OH2 bond lengths of 1.8 Å. (left), 2.4 Å (center), and 3.0 Å (right). The percent contributions from the Co 3d_z_2, water 2p_z_, and methyl 2p_z_ orbitals are noted. Red arrows indicate the donor and acceptor MOs involved in the α-band transition predicted for each model using TD-DFT.

Figure 8

TD-DFT/B3LYP predicted Abs spectra for QM/MM optimized MeCbi+ models with lower axial ligand bond lengths fixed at values ranging from 1.800 to 2.700 Å. All calculated spectra were red-shifted by 3000 cm−1 to facilitate comparison with the experimental spectroscopic data presented above.

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Spectroscopic studies of the corrinoid/iron-sulfur protein from Moorella thermoacetica - PubMed (original) (raw)