EVIDENCE OF HISTIDINE COORDINATION TO THE CATALYTIC FERROUS ION IN THE RING-CLEAVLNG 2,2',3-TRIHYDROXYBIPHENYL DIOXYGENASE FROM THE DIBENZOFURAN-DEGRADING BACTERIUM SPHINGOMONAS SP STRAIN RW1

The H-1 NMR spectra of an aromatic ring-cleaving extradiol dioxygenase , 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytica lly active reduced form of the monomeric enzyme (MW=32 kDa), three bro ad strongly downfield shifted signals were observed, two of which disa ppeared in D2O solution. Their shifts and linewidths are consistent wi th ring NH and meta-like protons of coordinated histidines. These sign als show strong sensitivity to the presence of the substrate. The oxid ized form of the enzyme shows no hyperfine shifted signals. It is sugg ested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This. is the first r eport of hyperfine shifted NMR signals being detected for an extradiol dioxygenase. (C) 1995 Academic Press, Inc.