Thiol-Disulfide Exchange Kinetics and Redox Potential of the Coenzyme M and Coenzyme B Heterodisulfide, an Electron Acceptor Coupled to Energy Conservation in Methanogenic Archaea.

Methanogenic and methanotrophic archaea play important roles in the global carbon cycle by interconverting CO2 and methane. To conserve energy from these metabolic pathways that happen close to the thermodynamic equilibrium, specific electron carriers have evolved to balance the redox potentials between key steps. Reduced ferredoxins required to activate CO2 are provided by energetical coupling to the reduction of the high-potential heterodisulfide (HDS) of coenzyme M (2-mercaptoethanesulfonate) and coenzyme B (7-mercaptoheptanoylthreonine phosphate). While the standard redox potential of this important HDS has been determined previously to be -143 mV (Tietze et al. 2003 DOI: 10.1002/cbic.200390053), we have measured thiol disulfide exchange kinetics and reassessed this value by equilibrating thiol-disulfide mixtures of coenzyme M, coenzyme B, and mercaptoethanol. We determined the redox potential of the HDS of coenzyme M and coenzyme B to be -16.4±1.7 mV relative to the reference thiol mercaptoethanol (E0 '=-264 mV). The resulting E0 ' values are -281 mV for the HDS, -271 mV for the homodisulfide of coenzyme M, and -270 mV for the homodisulfide of coenzyme B. We discuss the importance of these updated values for the physiology of methanogenic and methanotrophic archaea and their implications in terms of energy conservation.

Zero-quantum filtered pure shift TOCSY.

The high spectral resolution provided by the pure shift TOCSY experiment can be significantly improved by zero-quantum filtering which eliminates dispersive anti-phase contributions from the spectrum.

Conformational ensembles of flexible beta-turn mimetics in DMSO-d6.

Beta-turns play an important role in peptide and protein chemistry, biophysics, and bioinformatics. The aim of this research was to study short linear peptides that have a high propensity to form beta-turn structures in solution. In particular, we examined conformational ensembles of beta-turn forming peptides with a general sequence CBz-L-Ala-L-Xaa-Gly-L-Ala-OtBu. These tetrapeptides, APGA, A(4R)MePGA, and A(4S)MePGA, incorporate proline, (4R)-methylproline, and (4S)-methylproline, respectively, at the Xaa position. To determine the influence of the 4-methyl substituted prolines on the beta-turn populations, the NAMFIS (NMR analysis of molecular flexibility in solution) deconvolution analysis for these three peptides were performed in DMSO-d(6) solution. The NBO (natural bond orbital) method was employed to gain further insight into the results obtained from the NAMFIS analysis. The emphasis in the NBO analysis was to characterize remote intramolecular interactions that could influence the backbone-backbone interactions contributing to beta-turn stability. NAMFIS results indicate that the enantiospecific incorporation of the methyl substituent at the C(gamma) (C4) position of the proline residue can be used to selectively control the pyrrolidine ring puckering propensities and, consequently, the preferred varphi,psi angles associated with the proline residue in beta-turn forming peptides. The NAMFIS analyses show that the presence of (4S)-methylproline in A(4S)MePGA considerably increased the type II beta-turn population with respect to APGA and A(4R)MePGA. The NBO calculations suggest that this observation can be rationalized based on an n-->pi* interaction between the N-terminus alanine carbonyl oxygen and the proline carbonyl group. Several other interactions between remote orbitals in these peptides provide a more detailed explanation for the observed population distributions.

Importance of structural tightening, as opposed to partially bound States, in the determination of chemical shift changes at noncovalently bonded interfaces.

Two models (A and B) have been proposed to account for decreased downfield chemical shifts of a proton bound by noncovalent interactions at a ligand/antibiotic interface as the number of ligand/antibiotic interactions is decreased. In model A, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is, with a relatively large probability, broken, and not because it is longer. In model B, the proton involved in the noncovalent bond suffers a smaller downfield shift because the bond is longer, and not because it is, with a relatively large probability, broken. We show that model A cannot account for the chemical shift changes. Model B accounts for the process of positively cooperative binding, in which noncovalent bonds are reduced in length and thereby increase the stability of the organized state.