Hydrophobic modulation of heme properties in heme protein maquettes

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 f our-helix bundle protein maquette [H10H24-L61,L13F](2), here called [I6F13H 24](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectiv ely. The primary structure of each alpha -helix, alpha -SH, in [I6F13H24](2 ) is Ac-CGGGEI(6)WKL.(HEEFLKK)-E-10-L-13.FEELLKL.(HEERLKK)-E-24.L-CONH2. In our nomenclature, [I6F13H24] represents the disulfide-bridged di-alpha -he lical homodimer of this sequence. i.e.. (alpha -SS-alpha). and [I6F13H24](2 ) represents the dimeric four helix bundle composed of two di-alpha -helica l subunits, i.e., (alpha -SS-alpha)(2). We replaced the histidines at posit ions H24 in [I6F13H24](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I6F13I24](2), [I(6)F(13)A(24)](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 o ptimal amino acid replacement for H24 in the context of apo state conformat ional specificity. Furthermore, the novel second generation diheme [I6F13F2 4](2) maquette was related to the first generation diheme [H10A24](2) proto type, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential pat h in sequence space to evaluate the effects of conservative hydrophobic ami no acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (> 77% as determined from circular dichroism sp ectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H2O) > 1.8 kcal/mol), and possesses conformational specificity that NMR data indi cate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K-d1 value, tighter than 65 nM form ing 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 pepti de sequences are responsible for up to 10-fold changes in Kd values. The eq uilibrium 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 fro m -144 to -288 mV. An observed heme-heme electrostatic interaction (> 70 mV ) in the diheme state indicates a syn global topology of the di-alpha -heli cal monomers. The heme affinity and electrochemistry of the three H24 varia nts studied identify the tight binding sites (K-di and K-d2 values < 200 nM ) having the lower reduction midpoint potentials (E-m7.5 values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24 -L61.L13F](2), here called [I6F13H24](2). The results of this study illustrate that conservative hydrophobic amino ac id changes near the heme binding site can modulate the E-m by up to +/- 50 mV and the K-d by an order of magnitude. Furthermore, the effects of multip le single amino acid changes on E-m and K-d do not appear to be additive.