Proton nuclear magnetic resonance study of the solution distal histidine orientation in monomeric Chironomus thummi thummi cyanomet hemoglobins

Solution 1H‐NMR Structure of the Heme Cavity in the Low‐Affinity State for the Allosteric Monomeric Cyano‐Met Hemoglobins from Chironomus thummi thummi

“…3D), which identifies the C6H2-CyH part of Arg90(FG3). This is confirmed by the observation of NOEs from the previously assigned low-field hyperfine-shifted labile NCH, proton [13]. The CD loop makes a distal pocket contact with pyrrole D. A low-field hyperfine-shifted and strongly relaxed (T,=13 ms) single proton peak with NOE to 18CH3, 17Ha [15] and to a two-spin system (with detected COSY peaks) with upfield temperature intercept in the aromatic region, identifies the complete Phe38(CD1) ring.…”

“…NeH, yields R, = 0.41 50.03 nm with Eqn ( 2 ) [13], which is shorter than that observed for the orientation with His E7 in the pocket (0.48 nm) in the crystal structure [ll], indicating that His58(E7) is clearly oriented into the heme pocket, but not precisely as found in the disorder component in the cyanomet HbIII crystal structure with His E7 in the pocket. The G helix residues Gln96(G4), Leu97(G5), and PhelOO(G8) make contact with the heme at the 3-viny1, 3-vinyl and 7CH,, 3-vinyl and 7CH, %vinyl, respectively (Figs 3D, 4D).…”

“…The two COSY cross peaks with temperature-dependent shifts in cyanomet HbIII Arg61 (ElO), that correspond to those for Lys61(ElO) in cyanomet HbIV described above, were identified but did not exhibit detectable NOEs to the 12CH,. The His58(E7) side chain has been identified previously by the characteristic temperature-dependent shifted low-field ring NEH signal whose strong NOE to a sharp resolved singlet identifies the ring C6H [13]. The latter proton exhibits a NOESY cross peak to the Phe38(CD1) ring (Fig.…”

“…3, A, B and C, respectively, and the upfield portions in Fig. 4, A, B and C, respectively; the position of hyperfine-shifted labile protons for the native proteins, characterized in detail previously [13], are shown by vertical arrows. Representative NOESY data used to make assignments and to deduce structure for the two native Hb complexes are shown in Figs The NOESY spectrum was processed as described in Fig.…”

“…His87(F8) with low-field hyperfine-shifted NH, CaH and C m peaks; the NH shows the expected strong NOE when the His87(F8) ring N6H signal (Tl=30 ms) is saturated [13]. The F helix-heme contacts Phe83(F4) C6Hs-8Ha ( Fig.…”