Mark J. Nelson

Design of a heme-binding four-helix bundle

The design and characterization of two synthetic peptides that self-assemble into heme-binding proteins are described. The peptides are intended to fold into a four-helix bundle and bind a single heme parallel to the helices in the bundle core using two histidine side chains as ligands. Both proteins bind a single heme in the binding pocket. In one protein there are comparable amounts of low- and high-spin hemes, while in the other low-spin heme predominates. In both proteins, the EPR spectra of the low-spin heme indicate bis-imidazole ligation. The results illustrate that subtle differences in packing, binding pocket flexibility, and ligand orientation can significantly influence the characteristics of functionalized peptides

Effects of substrates (methyl isocyanide, C2H2) and inhibitor (CO) on resting-state wild-type and NifV(-)Klebsiella pneumoniae MoFe proteins.

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV(-)proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactors geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.

The Mechanism of Lipoxygenases

Lipoxygenases catalyze the biosynthesis of fatty acid hydroperoxides from polyunsaturated fatty acids and fatty acid esters. In mammals the fatty acid hydroperoxides are substrates for the pathways that lead to leukotrienes and lipoxins, potent messengers that are involved in the inflammatory response (Samuelsson et al., 1987; Wasserman et al., 1991). Consequently, lipoxygenase inhibitors have been a major goal of the pharmaceutical industry as potential drugs against, e.g., arthritis and asthma (Batt, 1992; McMillan and Walker, 1992). The fatty acid hydroperoxides themselves may play a role in a variety of phenomena, including cell maturation and the development of atherosclerosis (see Chapter 10) (Schewe and Kuhn, 1991). In plants the role of lipoxygenase is less well understood; here the fatty acid hydroperoxide is a substrate for pathways that lead to production of species such as jasmonic and traumatic acids that appear to be involved in events as diverse as development and growth regulation, wound response, and pest resistance (Gardner, 1991; Hildebrand and Grayburn, 1991; Siedow, 1991). The most familiar role of these fatty acid hydroperoxides, however, is as substrates for the production of cis-3-hexenol and trans-2-hexenal, the species responsible for the odor of new-mown grass.