Analysis of Fe(III) Heme Binding to Cysteine-Containing Heme-Regulatory Motifs in Proteins

in: ACS Chemical Biology (2013)
Galler, Kerstin; Neugebauer, Ute; Popp, Jürgen; Heinemann, Stefan H.; Kühl, Toni; Wißbrock, Amelie; Goradia, Nishit; Sahoo, Nirakar; Ohlenschläger, Oliver; Imhof, Diana
Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420−430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420−430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV−vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CPpeptide- heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzyme’s activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.

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