Very Early Reaction Intermediates Detected by Microsecond Time Scale Kinetics of Cytochrome cd1-catalyzed Reduction of Nitrite

Abstract

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 (ϳ100 s) 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.

Observation of fast release of NO from ferrous d1 haem allows formulation of a unified reaction mechanism for cytochrome cd1 nitrite reductases

“…-NO derivative of P. pantotrophus cytochrome cd 1 is a very long-lived species in the absence of excess reductant [14,30]; it also agrees with previous ultra-fast (µs) kinetic studies on the same enzyme showing that intramolecular c haem to d 1 haem electron transfer triggered product release [31]. All available data indicate that P. pantotrophus cytochrome cd 1 only works efficiently in the presence of substrate and electron donors [18,30,32,33], i.e.…”

Design of turbulent tangential micro-mixers that mix liquids on the nanosecond time scale

“…The great majority of proteins are stable at these pressures [47], further indicated by the general experience that these HPLC pumps can be used for purification of native active proteins and enzymes by column chromatography. In our freeze-quench experiments using the same HPLC setup and stainless-steel mixer described here, we have never encountered denaturation problems and the rate constants determined at 20 to 40 MPa by us [28,43,44,48,49] are similar to those determined by others working at ambient pressures [5,6,8,12]. In general, oligomeric proteins start to dissociate at pressures of 100 to 200 MPa, and monomeric proteins denature at approximately four times higher pressures [47].…”

The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase

“…These conclusions on Pa-cd 1 NiR are also supported by the experiments carried out on the enzyme from P. pantotrophus suggesting that intramolecular c-haem to d 1 -haem electron transfer triggers product release [43]; indeed it has been shown that P. pantotrophus d 1 Fe(III)-NO complex is a very long-lived species in the absence of excess reductant [44,45]. Therefore also P. pantotrophus cd 1 NiR works efficiently only in the presence of substrate and electron donor proteins [16,44,46], i.e.…”