Hydrophobic Modulation of Heme Properties in Heme Protein Maquettes

• Published in: Biochemistry (Easton) 2001-09, Vol.40 (35), p.10550-10561
• Main Authors: Gibney, Brian R, Huang, Steve S, Skalicky, Jack J, Fuentes, Ernesto J, Wand, A. Joshua, Dutton, P. Leslie
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Circular Dichroism; Heme - chemistry; Hemeproteins - chemistry; Histidine - chemistry; Molecular Sequence Data; Molecular Weight; Protein Conformation; Spectrophotometry, Ultraviolet; Thermodynamics; Water - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi002806h

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F]2, here called [I6F13H24]2 for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each α-helix, α-SH, in [I6F13H24]2 is Ac-CGGGEI6WKL·H10EEF13LKK·FEELLKL·H24EERLKK·L-CONH2. In our nomenclature, [I6F13H24] represents the disulfide-bridged di-α-helical homodimer of this sequence, i.e., (α-SS-α), and [I6F13H24]2 represents the dimeric four helix bundle composed of two di-α-helical subunits, i.e., (α-SS-α)2. We replaced the histidines at positions H24 in [I6F13H24]2 with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I6F13I24]2, [I6F13A24]2, and [I6F13F24]2, are distinguished from the tetraheme binding parent peptide, [I6F13H24]2, by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I6F13F24]2 maquette was related to the first generation diheme [H10A24]2 prototype, [L6L13A24]2 in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (−ΔG H 2 O >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K d1 value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K d2 in the range of 35−800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K d values. The equilibrium reduction midpoint potential (E m7.5) determined in the monoheme state ranges from −156 to −210 mV vs SHE and in the diheme state ranges from −144 to −288 mV. An observed heme−heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global top