MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 90

nucleotide hydrolysis may serve as a regulator of conformational switching. In contrast,
only a small conformational change in A1 was detected. Experiments with mutant A2
deleted of residue Leu 127 indicated a key role of this group in supporting mechanically
productive ATP hydrolysis. Binding of ATP or ADP to the Fe-protein stabilizes its
oxidizing state and lowers the redox potential by -0.10 V (Watt et al., 1986).
The Asp39 group in Av2, which is located in the vicinity of of ATP, was
genetically replaced for asparagine and three forms of the proteins were isolated: wild-
type homodimeric [Asp39/Asp39], mutated heterodimeric [Asp39/Asn 39], and
homodimeric [Asn39/Asp39] forms (Chan et al., 2000). The assessment of the MgATP
binding-induced conformational changes in Av2 indicated that the nucleotide effect
weakens in succession: [Asp39/Asp39] > [Asp39/Asn 39] > [Asn39/Asp39]. In this
expression, the relative substrate reduction activity of the protein forms was found to be
Thus, present results reveal that carboxylic residues of the Asp39 groups in
native Av2 play a key role in protein activity and only simultaneous action of both
groups maintains the high rate of the substrate reduction.
It was shown that the product of ATP hydrolysis, MgADP, remains bound on the
enzyme for the time necessary for the formation of (Syrtsova et al., 1988).
The rate of MgADP elimination from the nitrogenase molecule at the stage of transfer of
the first electron is low: the first MgADP molecule separates from nitrogenase with
and the second molecule does with It has been established in
stopped-flow calorimetry experiments at 60 °C and at pH 7.0 that a proton is released
from the ATP-Kpl.Kp2 complex before the electron transfer (Thorneley et
al., 1989; Thorneley and Dean, 2000). is found to be liberated after the intramolecular
electron transfer during ATP hydrolysis by nitrogenase (Lowe et al., 1995).
In the case of Av2 mutant (Leu 127 deleted, the midpoint potential for the
transition is changed from by -0.420 eV for the
free protein to -0.620 eV for its complex with A1 (Lanzilotta and Seefeldt, 1997). Only
a slight shift by -0.08 eV and no marked shift were observed in potentials of P-cluster
and FeMoco, respectively. In the presence of dithionate, the second electron is not
transferred to Av2 without MgATP, but the state was observed using Ti (III) as
a reductant (Nyborg et al., 2000). The energetic profile of the nitrogenase reaction,
presented in Fig. 3.7, indicates that formation of reaction plausible intermediates is
thermodynamically forbidden without utilization energy of ATP hydrolysis
(Likhtenshtein, 1979, 1988a; Likhtenshtein and Shilov, 1976; Syrtsova and Timofeeva,
2001).
Avaible experimental structural and kinetics data and energetic considerations
indicate two plausible roles of ATP in the nitrogenase reduction: a) the triggering of
electron transfer from iron protein to iron-molybdenum protein (Howard and Rees, 1994;
Rees and Howard, 2000) and the strengthening reducing power of the enzyme catalytic
redox centers (Likhtenshtein and Shilov, 1977, Likhenshtein 1988a, Syrtsova and
Timofeeva, 2001; see also Section 6.1.4).
Taking into consideration the X-ray structural model of the Fe-protein complex with
we can discuss a possible mechanism for utilization of the ATP hydrolysis
energy. According to our model, the protein undergoes substantial structural change at