ELECTROSTATIC MODIFICATION OF THE ACTIVE-SITE OF MYOGLOBIN - CHARACTERIZATION OF THE PROXIMAL SER92ASP VARIANT

The structural and functional consequences of the introduction of a ne gatively charged amino acid into the active site of horse heart myoglo bin have been investigated by replacement of the proximal Ser92 residu e (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxi ma of various ferrous and ferric derivatives and low-temperature EPR s pectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the var iant. H-1-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92As p variant is consistent with this finding and with a pK(a) = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type prot ein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in confo rmation of the proximal (His93) or distal (Wat156) heme ligands. H-1-N MR spectroscopy of the metMbCN form of the protein indicates that a sl ight rotation of the proximal His93 ligand has occurred in this deriva tive. Resonance Raman experiments indicate increased conformational he terogeneity in the proximal pocket of the variant. Failure to detect e lectron density for the Asp residue in the X-ray diffraction map of th e variant protein and high average thermal factors for the pyrrole A p ropionate substituent are consistent with this observation. The varian t exhibits novel pH-dependent behavior in the metMb form, as shown by H-1-NMR spectroscopy, and provides evidence for a heme-linked titratab le group with a pK(a) of 5.4 in this derivative. The metMbCN and deoxy Mb derivatives also exhibit pH-dependent behavior, with pK(a)s of 5.60 +/- 0.07 and 6.60 +/- 0.07, respectively, compared to the wild-type v alues of 5.4 +/- 0.04 and 5.8 +/- 0.1. The heme-linked ionizable group is proposed to be His97 in all three derivatives. The reduction poten tial of the variant is 72 +/- 2 mV vs SHE [25.0 degrees C, mu = 0.10 M (phosphate), pH 6.0], an increase of 8 mV over the wild-type value. T he possible influence of a number of variables on the magnitude of the reduction potential in myoglobin and other heme proteins is discussed .