Unusual Heme Iron-Lipid Acyl Chain Coordination in Escherichia coli Flavohemoglobin

Abstract

Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 A in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 A (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 A resolution obtained in the present work. Thus, the contributions at 2.7 A distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid.

Flavohemoglobin: Structure and reactivity

“…In agreement with the crystallographic data, optical absorption (both in solution and in crystals) and resonance Raman spectroscopy point out unambiguously that the lipid-free ferric derivative is a pentacoordinate species, and the Leu57(E11) residue does not establish a bonding interaction with the ferric iron. In contrast, the formation of a hexacoordinate derivative is clearly demonstrated upon lipid binding to the ferric HMP protein by the low frequency shift of the core size marker bands (22). The appearance of a hexacoordinate high spin species is accompanied by an unusually intense band at (23).…”

Diversity of Globin Function: Enzymatic, Transport, Storage, and Sensing

“…coli FHb is inactive toward peroxidase substrates, and the ferric FHb does not react with H 2 O 2 . However, the latter binds unsaturated or cyclopropanated fatty acids and acts as an alkylhydroperoxide reductase with NADH as electron donor, catalyzing the reduction of alkylhydroperoxides to the corresponding alcohols (30). These results suggest that FHb may be involved in the repair of oxidatively damaged lipid membranes.…”

Unusual proximal heme pocket geometry in the deoxygenated Thermobifida fusca: A combined spectroscopic investigation

“…An accurate structural characterization has been obtained on metal ionic solutions [12], different heme models and heme containing proteins [13][14][15], showing that a combined analysis of the EXAFS and XANES regions of the spectrum is able to provide unambiguous details on the local structure in solution around the metal center.…”

“…Previous investigations have shown that for a quantitative EXAFS analysis of hemeproteins MS fourbody terms have to be accounted for [13][14][15][16]20]. The inclusion of these higher order contributions is essential to obtain a good agreement between theoretical and experimental data.…”

“…A thorough description of the data analysis procedure and all the relevant parameters used in the minimization is given in Ref. [13].…”