The final step in the biosynthesis of the plant
hormone ethylene is catalyzed by the non-heme ironcontaining
enzyme 1-aminocyclopropane-1-carboxylic
acid (ACC) oxidase (ACCO). ACC is oxidized at the
expense of O2 to yield ethylene, HCN, CO2, and two
waters. Continuous turnover of ACCO requires the
presence of ascorbate and HCO3
) (or an alternative
form), but the roles played by these reagents, the order
of substrate addition, and the mechanism of oxygen
activation are controversial. Here these issues are addressed
by development of the first functional single
turnover system for ACCO. It is shown that 0.35 mol
ethylene/mol Fe(II)ACCO is produced when the enzyme
is combined with ACC and O2 in the presence of HCO3
)
but in the absence of ascorbate. Thus, ascorbate is not
required for O2 activation or product formation. Little
product is observed in the absence of HCO3
)
, demonstrating
the essential role of this reagent. By monitoring
the EPR spectrum of the sample during single turnover,
it is shown that the active site Fe(II) oxidizes to Fe(III)
during the single turnover. This suggests that the electrons
needed for catalysis can be derived from a fraction
of the initial Fe(II)ACCO instead of ascorbate. Addition
of ascorbate at 10% of its Km value significantly accelerates
both iron oxidation and ethylene formation,
suggesting a novel high-affinity effector role for this reagent.
This role can be partially mimicked by a nonredox-active
ascorbate analog. A mechanism is
proposed that begins with ACC and O2 binding, iron
oxidation, and one-electron reduction to form a peroxy
intermediate. Breakdown of this intermediate, perhaps
by HCO3
)
-mediated proton transfer, is proposed
to yield a high-valent iron species, which is the true
oxidizing reagent for the bound ACC.