Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd 1 nitrite reductase: regulation of the NO-bound end product

Abstract

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.

A new CuZ active form in the catalytic reduction of N2O by nitrous oxide reductase from Pseudomonas nautica

“…Cytochrome c-552 was used in mediated electrochemical experiments for the reduction of nitrite by Ps. nautica cytochrome cd 1 nitrite reductase [21] and a similar rate constant was calculated in that study [k = (4.1 ± 0.1) Step 3…”

“…The electrochemical behaviour of this cytochrome has been well characterized [19], and it has already been shown to be the electron donor of other enzymes isolated from Ps. nautica, cytochrome c peroxidase [20] and cytochrome cd 1 nitrite reductase [21]. In the latter case the electron transfer reaction was also investigated using electrochemical techniques.…”

Solvent Stress Response of the Denitrifying Bacterium “ Aromatoleum aromaticum ” Strain EbN1

“…One may speculate that aromatic compounds could interfere with electron flow to or within NirS, e.g., by inhibiting NirS dimerization or the NirS-cytochrome c interaction. An impaired electron flow was reported to result in NirS inactivation by irreversible NO binding in Pseudomonas nautica (29). Hence, an increased NirS concentration might compensate for solvent-induced NirS inactivation in strain EbN1.…”

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

“…The theory describing such a mechanism for diffusioncontrolled processes was developed by Nicholson and Shain [36] and applied to several kinetics studies of reactions between mediators and redox proteins [37] (and references therein). However, since the catalytic current observed in our experimental conditions is independent of the scan rate for v < 100 mV s -1 , thin-layer theory must be considered following Laviron's treatment for diffusionless electrochemical systems [31].…”

“…This value compares well with other intermolecular rate constants determined using cyclic voltammetry. Two examples are the electron transfer from Achromobacter cycloclastes PAz to its electron donor, nitrite reductase, that was determined to have a k value of 7.3 · 10 5 M -1 s -1 [40], and the complex between cytochrome c 552 and cytochrome cd 1 nitrite reductase isolated from Pseudomonas nautica, with a k value of 2.8 · 10 5 M -1 s -1 [37].…”