J Biol Chem, Vol. 274, Issue 4, 2029-2037, January 22, 1999

Reactions of Sperm Whale Myoglobin with Hydrogen Peroxide
EFFECTS OF DISTAL POCKET MUTATIONS ON THE FORMATION AND STABILITY OF THE FERRYL INTERMEDIATE

From the Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

Raymond F. Eich, and John S. Olson

From the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892

Robert E. Cashon

From the Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, Maine 04469-5735

Distal pocket mutants of sperm whale oxymyoglobin (oxy-Mb) were reacted with a 2.5-fold excess of hydrogen peroxide (HOOH) in phosphate buffer at pH 7.0, 37 °C. We describe a mechanism composed of three distinct steps: 1) initial oxidation of oxy- to ferryl-Mb, 2) autoreduction of the ferryl intermediate to ferric metmyoglobin (metMb), and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle. Mutation of Leu-29(B10) to Phe slows the initial oxidation reaction 3-fold but has little effect on the rate of ferryl reduction to ferric met-aquo-myoglobin. In contrast, the Val-68(E11) to Phe mutation causes a small, 60% increase in the initial oxidation reaction and a much larger 2.5-fold increase in the rate of autoreduction. Double insertion of Phe at both the B10- and E11-positions (L29F/V68F) produces a mutant with oxidation characteristics of both single mutants, slow initial oxidation, and rapid autoreduction, but an extraordinarily high affinity for O₂. Replacing His-64(E7) with Gln produces 3-4-fold increases in both processes. Combining the mutation H64Q with L29F results in a myoglobin with enhanced resistance to metMb formation in the absence of antioxidant enzymes (i.e. catalase and superoxide dismutase) due to its own high pseudoperoxidase activity, which rapidly removes any HOOH produced in the initial stages of autoxidation. This double substitution occurs naturally in the myoglobin of Asian elephants, and similar multiple replacements have been used to reduce selectively the rate of nitric oxide (NO)-induced oxidation of both recombinant MbO₂ and HbO₂ blood substitute prototypes without altering O₂ affinity.