Abstract
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 degreesC. 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.
Highlights
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, and 3) reaction of metMb with an additional HOOH molecule to regenerate the ferryl intermediate creating a pseudoperoxidase catalytic cycle
Reaction of HOOH with the Oxy Forms of Recombinant Myoglobins (His-64)—Spectral changes accompanying the addition of HOOH to sperm whale oxy-Mb in a molar ratio of 1 heme:2.5 HOOH are shown in Fig. 1 and are similar to those reported by Shikama and co-workers [32, 33]
Mechanistic analysis of the reaction of hemoglobins/myoglobins with HOOH has revealed the formation of a higher oxidation state, the ferryl heme iron (Fe4ϩ), which can be detected by optical spectroscopy [30, 32, 34], and a globin-associated free radical, which can only be detected by EPR spectroscopy [14, 37]
Summary
Myoglobin Mutagenesis—Recombinant sperm whale myoglobins were constructed, expressed, and purified as described previously by Carver et al [25], Springer et al [26], and Egeberg et al [27], using the SCHEME 1 recombinant gene constructed by Springer and Silgar [28]. The first filtration step (high pH, high salt) was done in 50 mM phosphate (pH 8.3), 1 M NaCl. The following step (neutral pH, low salt) was done in 50 mM phosphate (pH 7.0) with no added NaCl. Ferryl-Mb was prepared by treating oxy-Mb with a 5-fold molar excess of HOOH in 50 mM phosphate buffer, pH 7.0. Initial multicomponent analysis of myoglobin oxidation products was performed according to Whitburn [31] in order to calculate the percentages of oxy-Mb, metMb, and ferryl-Mb at successive stages of the oxidation. Independent estimation of these intermediates along the reaction coordinates and estimation of the rate constants of the interconversions were performed as described below.
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