Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd₁ nitrite reductase: Regulation of the NO-bound end product

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 c_553(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 c_ox) is a second-order reaction with a rate constant (k₂) of 4.1±0.1×10⁵ 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 d_1ox. 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 d_1ox 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±1.1×10⁴ 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.