MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 86

In the presence of dithionate, FeMoco exposes the S= 3/2 ESR signal (Münck et al.,
1975); Lee et al., 1997). The transition requires the potential
(Pierick et al., 1993). The reduced P-cluster of Av2 transfers at least one
electron to to form the ESR silent "super-reduced" FeMoco,
(Ohrme-Johnson et al., 1972; Münck et al., 1975). A combination of cyclic
voltampermetry, potentiometry and ESR spectroscopy has allowed the observation in
isolated FeMoco of two redox transitions with essentially different potentials,
and (Newton et al., 1996). The first transition of FeMoco is from
oxidize state to semireduced state of FeMoco and the second transition is related to the
substrate-reducing

state in FeMoco during the nitrogenase reaction turnover.

Independently, for the redox pair the was estimated as

about -1.0 V (Syrtsova and Timofeeva, 2001).
The X-ray structural model of FeMoco indicates that FeMoco is completely buried in
the protein matrix approximately 10

from the surface, but a channel is formed at the
interstice between two of the protein subunits. According to molecular modeling
(Durrant, 2001), the Arg-bl05 side-chain nitrogen in Kp

Å

and water molecules of the
inner region act as a proton conductor from the bulk water to the homocitrate via the
Grotthuss mechanism. Other possible ways for proton transfer to a sulfur ligand of the
Kp

1

1 FeMoco are via a chain of Tyr 194 and His 360-His

is too high to transfer H + in Because the tyrosine acidity constant in proteins

the neutral pH, the hystidine channel looks more rational.
ESR, Mossbauer and X-ray absorbtion spectroscopy (K-edge EXAFS measurements)
studies have shown that Mo and V in the FeMo and FeV proteins are present in
analogous cofactor centers and have close ligand geometry (Harvey et al., 1990; Eady,
1996; Lei et al., 2000). The FeV- cofactor extracted from A. chroococcum was found to
be similar but not identical to FeMoco. Indirect evidence from genetic data and some
physical experiments suggests the structure of and P-clusters in Fe nitrogenase,
which contains only Fe centers, is also similar to those in Mo and V nitrogenases. (Eady,
1996;,Peters, 1999). ESR analysis shows the absence of signals related to S = 3/2 in Fe
nitrogenase which is typical of FeMo- and FeV cofactors. Maximum specific activity
(nmole of of protein/min) of nitrogenases isolated from different
microorganisms was found to be 1040, 660 and 350 units for FeMo-, FeV- and FeFe
proteins, correspondingly (Eady, 1996).

3.1.3. KINETICS AND MECHANISM OF THE NITROGENASE REACTION

The basic mechanism of nitrogenase with the use of dithionate as an electron donor for
the iron protein involves the following steps (Thorneley and Lowe, 1985; Likhtenshtein,
1988a; Burgess and Lowe, 1996; Smith, 1999; Seefeldt and Dean, 1997; Rees and
Howard, 2000; Syrtsova and Timofeeva, 2001): 1) reduction of Fe-protein with
flavodoxin or dithionate and attachment of two ATP molecules to the protein, 2)
formation of a complex between the reduced FeP with two bound ATP molecules and
FeMo-protein, 3) electron transfer between the reduced cluster of FeP to the P-
cluster of FeMoP coupled to the ATP hydrolysis, 4) electron transfer from P-cluster to