Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor.
Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.
Popović-Bijelić
Ana
A
Department of Biochemistry and Biophysics, Stockholm University, S-10691 Stockholm, Sweden.
Voevodskaya
Nina
N
Domkin
Vladimir
V
Thelander
Lars
L
Gräslund
Astrid
A
D000818:Animals; D002692:Chlamydia trachomatis / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008345:Manganese; D007501:Iron; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit