<i>In vitro</i>
            evolution of horse heart myoglobin to increase peroxidase activity

Wan, Lianglu; Twitchett, Mark B.; Eltis, Lindsay D.; Mauk, A. Grant; Smith, Michael B.

doi:10.1073/pnas.95.22.12825

Cited by 104 publications

(87 citation statements)

References 48 publications

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“…1), which significantly lowers the pK a values of catalytic His and Arg distal residues (66,67). This idea is further strengthened by the following: (i) the evidence that site-directed mutants of horse heart Mb (Thr39Ile, Lys45Asp, Phe46Leu, and Ile107Phe) and sperm whale Mb (Thr67Arg and Thr67Arg/ Ser92Asp) display a significant increase of the peroxidase activity (25,68,69), and (ii) site-directed mutants of cytochrome c peroxidase (His175Gln, His175Glu, and His175Cys) and horseradish peroxidase (Arg38Leu, His42Glu, His42Gln) show a substantial decrease of the peroxidase activity (70)(71)(72).…”

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confidence: 55%

Catalytic peroxidation of nitrogen monoxide and peroxynitrite by globins

et al. 2008

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SummaryGlobins are generally considered as carriers of diatomic gaseous ligands (e.g., O 2 and NO) in metazoa. Recently, the (pseudo-)enzymatic activity of globins towards reactive nitrogen and oxygen species has been elucidated. In particular, some globins (e.g., hemoglobin and myoglobin) catalyze the enzymatic scavenging of NO and peroxynitrite in the presence of H 2 O 2 . Indeed, H 2 O 2 oxidizes some globins leading to the formation of water and of the heme-protein ferryl derivative, which, in turn, oxidizes NO and peroxynitrite leading to the formation of the globin ferric species, NO 2 2 , and NO 3 2 . Here, we hypothesize that NO, peroxynitrite, and H 2 O 2 are co-substrates for the peroxidase activity of some globins, this catalytic activity was reported in 1900 for the first time, even though the substrates have never been identified firmly up to now. The hemoglobin (Hb) superfamily includes several hemeproteins, generally referred to as globins, which are found in all kingdoms of living organisms (1, 2). Globin functions have been the subject of active debate, in addition to dioxygen transport and storage. Several functions have been proposed recently, including control of nitrogen monoxide levels, O 2 sensing, and dehaloperoxidase activity (3-15).Globins share physical, spectroscopic, and chemical similarities with peroxidases (16,17). In fact, as demonstrated first in 1900 (18), Hb reacts readily with hydrogen peroxide (H 2 O 2 ). In 1923, the peroxidase activity of Hb has been reported (19), and in 1938, the modulation of the peroxidase activity of Hb by haptoglobin has been demonstrated (20). The reaction of myoglobin (Mb) with H 2 O 2 , on the other hand, apparently was not considered until 1952 (21), and the ability of Mb to catalyze peroxide oxidation of substrates was not reported until 1955 (22). Upon reaction with H 2 O 2 , Mb and Hb form the cytotoxic ferryl derivative (heme-Fe(IV)¼ ¼O), which is similar to compound II formed by peroxidases (23, 24). Heme-Fe(IV)¼ ¼O is able to oxidize a wide range of reducing substrates, such as phenols and aromatic amines, even though substrate peroxidation by Hb and Mb is far less efficient than that of peroxidases (24, 25), ruling out the possibility that the potential peroxidase activity of Hb and Mb is exerted on this class of substrates under normal conditions.Here, we hypothesize that the capability of some globins (e.g., Hb and Mb) to form a compound II-like species under oxidative stress may be actually exploited to avoid the building up of NO and peroxynitrite, 1 which can be then identified as the 'true' substrates for the peroxidase activity of Hb and Mb.Heme-proteins share the ability of detoxifying nitrogen reactive species, for example, NO. Even though leukocyte peroxi- 1 The term peroxynitrite is used in the text to refer generically to both ONOO 2 and its conjugated acid HOONO (38).

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confidence: 55%

Catalytic peroxidation of nitrogen monoxide and peroxynitrite by globins

et al. 2008

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“…42), and considerable interest has arisen in recent years in increasing this activity through the application of protein engineering technology (14,42,43). Although this objective was not a goal of the current work, it is noteworthy that amino acid substitutions introduced in the two variants studied here increase the peroxidase activity Ϸ3-fold and that this increase arises primarily through an increased rate of formation of the presumed compound I intermediate (k 1 , Table 3).…”

Section: Discussionmentioning

confidence: 91%

“…As part of our work to manipulate the peroxidase activity of horse heart myoglobin (Mb) (14,15), we have prepared a Mb variant in which two surface residues near the heme 6-propionate group have been replaced in an effort to introduce a metal ion binding site similar to that observed in the structure of manganese peroxidase. The effects of these substitutions and the resulting metal ion binding on selected functional properties of the variant have been evaluated, the nature of Mn 2ϩ binding to this site has been studied, and the structure of the Mn 2ϩ -Mb complex has been determined by x-ray crystallography.…”

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confidence: 99%

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Hunter

Maurus²,

Mauk³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1 H NMR spectroscopy indicates that Mn 2؉ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn 2؉ is 1.28(4) ؋ 10 4 M ؊1 (pH 6.96, ionic strength I ‫؍‬ 17.2 M, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK a for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn 2؉ -peroxidase activity of the variant are both increased Ϸ3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn 2؉ . The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn 2؉ and Cd 2؉ . Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.

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“…In the current studies, we compared the activities of the de novo proteins to that of myoglobin, which has moderate peroxidase activity. [26][27][28] Like our de novo proteins, myoglobin is an a-helical heme protein. Moreover, like our de novo proteins-and in contrast to natural peroxidases-myoglobin was not evolutionarily selected for peroxidase activity.…”

Section: Kinetic Profiles Of Purified Proteinsmentioning

confidence: 99%

Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4‐helix bundle proteins

et al. 2009

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To probe the potential for enzymatic activity in unevolved amino acid sequence space, we created a combinatorial library of de novo 4-helix bundle proteins. This collection of novel proteins can be considered an ''artificial superfamily'' of helical bundles. The superfamily of 102-residue proteins was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. Sequences from the library were screened for a range of biological functions including heme binding and peroxidase, esterase, and lipase activities. Proteins exhibiting these functions were purified and characterized biochemically. The majority of de novo proteins from this superfamily bound the heme cofactor, and a sizable fraction of the proteins showed activity significantly above background for at least one of the tested enzymatic activities. Moreover, several of the designed 4-helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies reveal that de novo proteins-which have neither been designed for function, nor subjected to evolutionary pressure (either in vivo or in vitro)-can provide rudimentary activities and serve as a ''feedstock'' for evolution.

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In vitro evolution of horse heart myoglobin to increase peroxidase activity

Cited by 104 publications

References 48 publications

Catalytic peroxidation of nitrogen monoxide and peroxynitrite by globins

Catalytic peroxidation of nitrogen monoxide and peroxynitrite by globins

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4‐helix bundle proteins

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