Rapid-freeze-quench magnetic circular dichroism of intermediate X in ribonucleotide reductase: New structural insight

To elucidate the electronic structure of intermediate X in the oxygen activation reaction of the R2 subunit of ribonucleotide reductase, a protocol has been developed to perform magnetic circular dichroism (MCD) on a rapid-freeze-quench, strain free optical sample. RFQ-MCD data have been collected on intermediate X in the double mutant of R2, Y122/Y356F. While X has been reported to exhibit a broad absorption band at 365 nm, there are at least 10 electronic transitions observed at low-temperature MCD. From C₀/D₀ ratios, the transitions of X can be divided into three regions: 16000-22000 cm^-1 region involving spin-allowed ligand field transitions of the Fe(^IV), 23000-24000 cm^-1 region of spin-forbidden, spin-flip transitions on the Fe(^IV), and the charge transfer (CT) region from 26000 to 32000 cm^-1. The C₀/D₀ ratios from d → d and CT transitions strongly support significant Fe(^IV) character coupled into the paramagnetic center. Ligand field (spin-allowed d → d region) analysis allows the bis-μ-oxo and μ-oxo plus other monoanionic bridge possibilities for the structure of intermediate X to be distinguished, providing new insight into the molecular mechanism of the cluster formation in R2.