Protein film voltammetry of copper-containing nitrite reductase reveals reversible inactivation.
The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments were carried out to obtain detailed information about the catalytic cycle. The homotrimeric structure of the enzyme is reflected in a distribution of the heterogeneous electron-transfer rates around three main values. Otherwise, the properties and the mode of operation of the enzyme when it is adsorbed as a film on a pyrolytic graphite electrode are essentially unchanged compared to those of the free enzyme in solution. It was established that the reduced type-2 site exists in either an active or an inactive conformation with an interconversion rate of approximately 0.1 s(-1). The random sequential mechanism comprises two routes, one in which the type-2 site is reduced first and subsequently binds nitrite, which is then converted into NO, and another in which the oxidized type-2 site binds nitrite and then accepts an electron to produce NO. At high nitrite concentration, the second route prevails and internal electron transfer is rate-limiting. The midpoint potentials of both sites could be established under catalytic conditions. Binding of nitrite to the type-2 site does not affect the midpoint potential of the type-1 site, thereby excluding cooperativity between the two sites.
Wijma
Hein J
HJ
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.
Jeuken
Lars J C
LJ
Verbeet
Martin Ph
MP
Armstrong
Fraser A
FA
Canters
Gerard W
GW
D003300:Copper / Q000737:chemistry; D004563:Electrochemistry; D007700:Kinetics; D009572:Nitrite Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; D003300:Copper; D009572:Nitrite Reductases