O(2)-evolving chlorite dismutase as a tool for studying O(2)-utilizing enzymes.
The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas's modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O(2) at concentrations far exceeding 2 mM. This method was used to define the O(2) concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme's Mn(IV)/Fe(III) cofactor forms from a Mn(II)/Fe(II) complex and O(2) via a Mn(IV)/Fe(IV) intermediate, at effective O(2) concentrations as high as ~10 mM. With a more soluble receptor, myoglobin, an O(2) adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C-H-bond-cleaving Fe(IV)-oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo-Fe(2)(III/III) complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe(2)(III/IV) intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O(2) evolution permits a >5 mM "pulse" of O(2) to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O(2) addition steps.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
Laura M K
LM
Dassama
Timothy H
TH
Yosca
Denise A
DA
Conner
Michael H
MH
Lee
Béatrice
B
Blanc
Bennett R
BR
Streit
Michael T
MT
Green
Jennifer L
JL
DuBois
Carsten
C
Krebs
J Martin
JM
Bollinger
D001426:Bacterial Proteins / Q000737:chemistry*; D002692:Chlamydia trachomatis; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D012264:Ribonucleotide Reductases / Q000737:chemistry; D015204:Spectroscopy, Mossbauer
D001426:Bacterial Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D008345:Manganese; D010088:Oxidoreductases; C402794:chlorite dismutase; D012264:Ribonucleotide Reductases; D010100:Oxygen