Engineering peroxidase activity in myoglobin: the haem cavity structure and peroxide activation in the T67R/S92D mutant and its derivative reconstituted with protohaemin-l-histidine

Abstract

Atomic co-ordinates and structure factors for the T67R/S92D metMbCN mutant have been deposited with the Protein Data Bank, under accession codes 1h1x and r1h1xsf, respectively. Protein engineering and cofactor replacement have been employed as tools to introduce/modulate peroxidase activity in sperm whale Mb (myoglobin). Based on the rationale that haem peroxidase active sites are characterized by specific charged residues, the Mb haem crevice has been modified to host a haem-distalpropionate Arg residue and a proximal Asp, yielding the T67R/S92D Mb mutant. To code extra conformational mobility around the haem, and to increase the peroxidase catalytic efficiency, the T67R/S92D Mb mutant has been subsequently reconstituted with protohaem-L-histidine methyl ester, yielding a stable derivative, T67R/S92D Mb-H. The crystal structure of T67R/S92D cyano-metMb (1.4 A resolution; R factor, 0.12) highlights a regular haem-cyanide binding mode, and the role for the mutated residues in affecting the haem propionates as well as the neighbouring water structure. The conformational disorder of the haem propionate-7 is evidenced by the NMR spectrum of the mutant. Ligand-binding studies show that the iron(III) centres of T67R/S92D Mb, and especially of T67R/S92D Mb-H, exhibit higher affinity for azide and imidazole than wild-type Mb. In addition, both protein derivatives react faster than wild-type Mb with hydrogen peroxide, showing higher peroxidase-like activity towards phenolic substrates. The catalytic efficiency of T67R/S92D Mb-H in these reactions is the highest so far reported for Mb derivatives. A model for the protein-substrate interaction is deduced based on the crystal structure and on the NMR spectra of protein-phenol complexes.

Catalytic peroxidation of nitrogen monoxide and peroxynitrite by globins

“…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).…”

Engineering and Prosthetic‐Group Modification of Myoglobin: Peroxidase Activity, Chemical Stability and Unfolding Properties

“…In particular, we replaced the distal Thr67 with either Arg (T67R Mb) [46] or the more flexible Lys residue (T67 K Mb) [47] and obtained a double mutant where, in addition to the Thr67Arg mutation, the proximal Ser92 was substituted with an Asp residue (T67R/S92D Mb) ( Figure 3). [48] The mutants T67 K Mb and T67R/S92D Mb were also reconstituted with HMϪH, while with T67R Mb we could not obtain a stable derivative. The reconstituted sperm whale Mb derivatives will be indicated as WT MbϪH, T67 K MbϪH and T67R/S92D MbϪH.…”

Advances in Engineered Hemoproteins that Promote Biocatalysis

“…A series of reports have described the engineering of the myoglobin active site to induce peroxygenase (sulfoxidation and epoxidation) and peroxidase reactivity [4,9,[44][45][46][47][48]. Many mutants have been described, and some have showed increased reactivity.…”

“…In its native form, peroxidative reactivity of substrates is not very efficient even though oxidative intermediates are presumably formed from the reaction with peroxides. Lack of reactivity of the native form of myoglobin has been examined and many research studies have concluded that the active site of myoglobin lacks a substrate-binding site, which would prohibit oxidative intermediates to productively react with substrates [4,8,24,25,[44][45][46][47][48]. To this end, many studies have employed the use of myoglobin mutants to affect a change of reactivity by (a) opening up the active site to afford substrate binding or (b) changing amino acids at the active site to mimic structural aspects of other enzymes and their functions.…”

“…Teale described a simple acidification and heme extraction technique which allowed for generations of hemoprotein researchers to easily exchange cofactors and explore new possibilities for functionalities as shown in Figure 2 [1]. Many decades later the utility of replacing the native heme cofactor with heme analogs has emerged as a strategy to produce synthetic metalloenzymes to function as robust biocatalysts [2][3][4][5][6][7][8][9][10].…”