Site-specific replacement of Y356 with 3,4-dihydroxyphenylalanine in the beta2 subunit of E. coli ribonucleotide reductase.
E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits alpha2 and beta2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chemistry. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 A between alpha2 and beta2. Y356 in beta2 has been proposed to lie on this pathway. To test this model, intein technology has been used to make beta2 semi-synthetically in which Y356 is replaced with a DOPA-amino acid. Analysis of this mutant with alpha2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y356 lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asymmetric.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, 02139-4307, USA.
Mohammad R
MR
Seyedsayamdost
Joanne
J
Stubbe
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D004295:Dihydroxyphenylalanine / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D005609:Free Radicals; D013942:Thymine Nucleotides; D006153:Guanosine Diphosphate; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C024157:thymidine 5'-triphosphate