TY - JOUR

T1 - Intramolecular electron transfer in pseudomonas aeruginosa cd1 Nitrite reductase

T2 - Thermodynamics and kinetics

AU - Farver, Ole

AU - Brunori, Maurizio

AU - Cutruzzolà, Francesca

AU - Rinaldo, Serena

AU - Wherland, Scot

AU - Pecht, Israel

PY - 2009

Y1 - 2009

N2 - The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion-controlled bimolecular process, followed by unimolecular electron equilibration between the c and d(1) hemes (k(ET) = 4.3 s(-1) and K = 1.4 at 298 K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by approximately 30-fold) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control based on the available 3D structure of the wild-type and the H369A mutant, in the reduced and oxidized states.

AB - The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion-controlled bimolecular process, followed by unimolecular electron equilibration between the c and d(1) hemes (k(ET) = 4.3 s(-1) and K = 1.4 at 298 K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by approximately 30-fold) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control based on the available 3D structure of the wild-type and the H369A mutant, in the reduced and oxidized states.

KW - Cytochromes

KW - Electron Transport

KW - Heme

KW - Kinetics

KW - Mutation

KW - Nitrite Reductases

KW - Protein Conformation

KW - Pseudomonas aeruginosa

KW - Pulse Radiolysis

KW - Thermodynamics

U2 - 10.1016/j.bpj.2008.12.3937

DO - 10.1016/j.bpj.2008.12.3937

M3 - Journal article

C2 - 19348767

VL - 96

SP - 2849

EP - 2856

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 7

ER -