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

Abstract

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 CP-peptide-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.

A heme-binding domain controls regulation of ATP-dependent potassium channels

“…4A) compared with free hemin (λ max = 385 nm). Similar absorption patterns (λ max = 412-417 nm) have been reported for heme binding to peptides with a single cysteine residue (14) and to proteins with Cys/His coordination (Table S1). Heme binding to a control peptide B (LPFESSKKATGVQSKPINRKQPGRFALDNFE) in which Cys and His residues have been replaced did not show any evidence of heme binding under the same conditions.…”

“…In the case of ion channel regulation, it has recently emerged that there is a role for heme (12,(23)(24)(25)(26), but the observations are largely empirical, so that the molecular basis for the regulatory control within individual channel proteins has yet to be properly defined. A number of heme-responsive motifs (27) have been suggested to be involved: these include Cys/Pro (CP) motifs using thiolate ligation to the heme as in the P450s or Cys/His motifs (3,14). In the case of the Slo1 channels (12), a cytochrome c-like CXXCH motif has been implicated (12,28).…”

Regulation of intracellular heme trafficking revealed by subcellular reporters

“…A number of heme-binding interactions have been suggested, including Cys/Pro (CP) motifs or Cys/His motifs (66,(68)(69)(70).…”

Plasmodium falciparum Merozoite Surface Protein 3

“…To further characterize the nature of the heme-MSP3 complex, we first quantitatively measured heme binding by two independent methods (17) and found that MSP3 can bind 35-40 mol of hemin/mol of protein. Such multiple heme-binding sites present in a protein sequence are often attributed to high histidine content or due to the presence heme regulatory motifs (61)(62)(63). For example, histidine-rich protein II that has high histidine content can bind 35-50 mol of heme/mol of protein (17).…”