Abstract
R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine–valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.
Highlights
Di-metal carboxylate proteins from the ferritin-like superfamily use a dinuclear metal cofactor to reduce oxygen and catalyze a variety of one- or two-electron redox reactions from the resulting high-valent state of the metal cluster [1,2,3,4,5]
Mn/Fe cofactor is found in the R2 subunits of subclass Ic ribonucleotide reductases as well as a related group of proteins known as R2-like ligand-binding oxidases (R2lox) [7]
We have recently demonstrated that the tyrosine–valine ether cross-link in R2lox is broken by intense blue light, causing the metallated protein to turn purple because Y162 coordinates the metal ion in site 2 instead of E167, which is decarboxylated [30]
Summary
Di-metal carboxylate proteins from the ferritin-like superfamily use a dinuclear metal cofactor to reduce oxygen and catalyze a variety of one- or two-electron redox reactions from the resulting high-valent state of the metal cluster [1,2,3,4,5]. Mn/Fe cofactor is found in the R2 subunits of subclass Ic ribonucleotide reductases (denoted as R2c) as well as a related group of proteins known as R2-like ligand-binding oxidases (R2lox) [7]. Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides via a radical-initiated mechanism. The R2 subunit of class I RNRs uses its dinuclear metal cofactor to generate this radical, which is reversibly transferred to the. All three known types of di-metal carboxylate cofactors are found in class I RNR R2 proteins: the prototypical subclass Ia uses a diiron cofactor [9], class Ib and the recently proposed class Id utilize a dimanganese cofactor [10,11,12,13,14,15], whereas class Ic contains a heterodinuclear
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