Modulation of the conductance of a 2,2′-bipyridine-functionalized peptidic ion channel by Ni2+

An α-helical amphipathic peptide with the sequence H2N-(LSSLLSL)3-CONH2 was obtained by solid phase synthesis and a 2,2′-bipyridine was coupled to its N-terminus, which allows complexation of Ni2+. Complexation of the 2,2′-bipyridine residues was proven by UV/Vis spectroscopy. The peptide helices were inserted into lipid bilayers (nano black lipid membranes, nano-BLMs) that suspend the pores of porous alumina substrates with a pore diameter of 60 nm by applying a potential difference. From single channel recordings, we were able to distinguish four distinct conductance states, which we attribute to an increasing number of peptide helices participating in the conducting helix bundle. Addition of Ni2+ in micromolar concentrations altered the conductance behaviour of the formed ion channels in nano-BLMs considerably. The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni2+ results in a considerable confinement of the observed multiple conductance states. However, the conductance levels were independent of the presence of Ni2+. Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni2+ diminishes the frequency of channel events with larger open times

Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes

‘Radical SAM’ enzymes generate catalytic radicals by combining a 4Fe–4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 Å resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe–4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 Å) and a neighboring sulfur of the cluster (3.6 Å), allowing single electron transfer from the 4Fe–4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5′-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme