Metalloprotein electron transfer reactions: analysis of reactivity of horse heart cytochrome c with inorganic complexes.

The reactions of horse heart cytochrome c with Fe(ethylenediaminetetraacetate)2-, Co(1,10-phenanthroline)3(3+), Ru(NH3)6(2+), and Fe(CN)6(3-) have been analyzed within the formalism of the Marcus theory of outer-sphere electron transfer, including compensation for electrostatic interactions. Calculated protein self-exchange rate constants based on crossreactions are found to vary over three orders of magnitude, decreasing according to Fe(CN)6(3-) greater than Ru(NH3)6(2+) greater than Fe(EDTA)2-. The reactivity order suggests that the mechanism of electron transfer involves attack by the small molecule reagents near the most nearly exposed region of the heme; this attack is affected by electrostatic interactions with the positively charged protein, by hydrophobic interactions that permit reagent penetration of the protein surface, and by the availability of pi symmetry ligand (or extended metal) orbitals that can overlap with the pi redox orbitals of the heme group

Reductant-dependent electron distribution among redox sites of laccase.

Rhus laccase (monophenol monooxygenase, monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) an O2/H2O oxidoreductase containing four copper ions bound to three redox sites (type 1, type 2, and type 3 Cu pair), was titrated anaerobically with several reductants having various chemical and thermodynamic properties. The distribution of electron equivalents among the redox sites was found to be reductant dependent. When the data for titration by various reductants of the type 3 site were plotted against those of the type 1 site according to the Nernst formalism, the slope n varied from 2.0 to 1.0. The redox potential of the reductant's first oxidation step is qualitatively correlated with the value of n and is suggested as the factor that modulates the electron distribution. Such a behavior implies a nonequilibrium situation. A very good simulation of the data was provided by an analysis assuming a formally variable cooperativity between the two type 3 copper ions. This apparent variability is suggested to result from a process whereby sufficiently strong reductants induce a transition of the type 3 site from a cooperative two-electron acceptor to a pair of independent one-electron acceptors. This uncoupled state of the type 3 site is considered metastable. Other possible models were also investigated. Summarizing the available data, we conclude that the two-electron accepting behavior of the 330-nm chromophore is the exception rather than the rule

Intramolecular electron transfer in Pseudomonas aeruginosa cd1 nitrite reductase: thermodynamics and kinetics

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