Very early reaction intermediates detected by microsecond time scale kinetics of cytochrome cd1-catalyzed reduction of nitrite.
Paracoccus pantotrophus cytochrome cd(1) is a nitrite reductase found in the periplasm of many denitrifying bacteria. It catalyzes the reduction of nitrite to nitric oxide during the denitrification part of the biological nitrogen cycle. Previous studies of early millisecond intermediates in the nitrite reduction reaction have shown, by comparison with pH 7.0, that at the optimum pH, approximately pH 6, the earliest intermediates were lost in the dead time of the instrument. Access to early time points (approximately 100 micros) through use of an ultra-rapid mixing device has identified a spectroscopically novel intermediate, assigned as the Michaelis complex, formed from reaction of fully reduced enzyme with nitrite. Spectroscopic observation of the subsequent transformation of this species has provided data that demand reappraisal of the general belief that the two subunits of the enzyme function independently.
Sam
Katharine A
KA
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Strampraad
Marc J F
MJF
Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands.
de Vries
Simon
S
Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands.
Ferguson
Stuart J
SJ
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. Electronic address: stuart.ferguson@bioch.ox.ac.uk.
D003580:Cytochromes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009572:Nitrite Reductases / Q000737:chemistry*; D009573:Nitrites / Q000737:chemistry*; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000201:enzymology*; D033903:Periplasmic Proteins / Q000737:chemistry*; D013997:Time Factors
D003580:Cytochromes; D009573:Nitrites; D033903:Periplasmic Proteins; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming)