STRUCTURE/FUNCTION 81

B026 SPECTROSCOPIC AND ELECTROCHEMICAL EFFECTS OF CHARGED AMINO ACIDS IN THE HEME BINDING POCKET OF HORSE HEART MYOGLOBIN

Emma Llovd and A. Grant Mauk Protein Engineering Network of Centres of Excellence and Department of Biochemistry and Molecular Biology, University of British CoIumbia, Vancouver, British Columbia V6T 123 Canada

The effect of charged amino acids on the active site chemistry of heme proteins is a subject of continuing interest.‘s2 Although it is clear that _~

W-68 Ku-61

local electrostatics have a substantial influence on P CT

%

W-67

the redox chemistry and spectroscopic properties of the heme group, the specific interactions PC responsible for these effects are not well understood. To investigate these effects, charged His-93 amino acids were introduced into the active site of horse heart myoglobin (Mb) by site-directed mutagenesis. The hydrophobic residues Va167 and V68, located within the distal heme pocket, c

were replaced by arginine and histidine A&W Site Of HorSe Har( MyO@Obh

respectively (V67R and V68H), and S92, located adjacent to the proximal histidine ligand, was replaced by an aspartic acid residue (S92D).

The E, of the V67R mutant was found to be 119.6 f 0.55 mV vs. NHE (pH 6.0,25”C, p = 0.10 M (NaCl)) compared to a value of 64.3 f 0.1 mV for wild type Mb. In addition, the pK,, for titration of the distal water molecule [Fe(III)-H,O + Fe(III)-OH- + H’] has been determined to be 8.0 for V67R, compared to 8.9 for wild-type metMb. For the S92D variant, the reduction potential was 98.7 f 1.0 mV vs. NHE (pH 6.0,25”C, u = 0.10 M (NaCl)), and the pKs, for the distal water molecule was 9.0.

Examination of the electronic spectrum of the V67R and S92D variants has revealed features that differ substantially from that of wild- type Mb. For example, the spectrum of deoxyMb exhibits new absorption maxima at 625.5 nm for V67R Mb and 624.5 nm for S92D Mb that are not present in the spectrum of wild-type deoxyMb. The effect of the introduction of a histidine residue at position 68 is currently under investigation. (Supported by the Protein Engineering Network of Centres of Excellence.)

1. J. Eccles, B. Honig, Proc. Natl. Acad. Sci. USA 80 4959 (1983). 2. R. Varadarajan, T. E. &wert, H. B. Gray, S. G. Boxer Science 243 69

(1989).