Spectroscopic and electronic structure studies of intermediate X in ribonucleotide reductase R2 and two variants: a description of the FeIV-oxo bond in the FeIII-O-FeIV dimer.
Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the FeIII-FeIV active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the FeIII/FeIV active site, which displayed three FeIV d-d transitions between 16,700 and 22,600 cm(-1), two FeIV d-d spin-flip transitions between 23,500 and 24,300 cm(-1), and five oxo to FeIV and FeIII charge transfer (CT) transitions between 25,000 and 32,000 cm(-1). The FeIV d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-pi manifold. Furthermore, the FeIV d-pi splittings in the WT are too large to correlate with a bis-mu-oxo structure. The assignment of the FeIV d-d transitions in WT intermediate X best correlates with a bridged mu-oxo/mu-hydroxo [FeIII(mu-O)(mu-OH)FeIV] structure. The mu-oxo/mu-hydroxo core structure provides an important sigma/pi superexchange pathway, which is not present in the bis-mu-oxo structure, to promote facile electron transfer from Y122 to the remote FeIV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis.
Mitić
Natasa
N
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Clay
Michael D
MD
Saleh
Lana
L
Bollinger
J Martin
JM
Solomon
Edward I
EI
D000327:Adsorption; D000465:Algorithms; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D005290:Ferric Compounds / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry*; D007202:Indicators and Reagents; D007501:Iron / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D019265:Spectroscopy, Near-Infrared; D013696:Temperature
D005290:Ferric Compounds; D005296:Ferrous Compounds; D007202:Indicators and Reagents; D007501:Iron; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit