RESUMEN
Factors governing the efficacy of long-range electron relays in enzymes have been examined using protein film voltammetry in conjunction with site-directed mutagenesis. Investigations of the fumarate reductase from Escherichia coli, in which three Fe-S clusters relay electrons over more than 30 A, lead to the conclusion that varying the medial [4Fe-4S] cluster potential over a 100 mV range does not have a significant effect on the inherent kinetics of electron transfer to and from the active-site flavin. The results support a proposal that the reduction potential of an individual electron relay site in a multicentered enzyme is not a strong determinant of activity; instead, as deduced from the potential dependence of catalytic electron transfer, electron flow through the intramolecular relay is rapid and reversible, and even uphill steps do not limit the catalytic rate.
Asunto(s)
Escherichia coli/enzimología , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Succinato Deshidrogenasa/química , Succinato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Electroquímica , Flavinas/química , Flavinas/metabolismo , Proteínas Hierro-Azufre/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Missense , Oxidación-Reducción , Alineación de Secuencia , Relación Estructura-Actividad , Succinato Deshidrogenasa/genéticaRESUMEN
The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli.
Asunto(s)
Proteínas de Escherichia coli/química , Nitrato Reductasas/química , Nitratos/química , Azidas/química , Sitios de Unión , Catálisis , Cloruros/química , Citoplasma/enzimología , Transporte de Electrón , Inhibidores Enzimáticos/química , Concentración de Iones de Hidrógeno , Molibdeno/química , Nitrato-Reductasa , Nitratos/antagonistas & inhibidores , Nitritos/química , Oxidación-Reducción , Potenciometría , Tiocianatos/químicaRESUMEN
It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation.