MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 82

Since the time of Daniel Rutherford, who discovered molecular nitrogen about 200
years ago, this gas has served as an example of a very inert substance. Thus, the
mechanism of the relatively fast reduction of in the nitrogenase active site with
turnover about appears as a mysterious and challenging problem not only for
biochemists but for chemists as well.
Recent developments in this important field have been reviewed in the last decade
(Burgess and Lowe, 1996; Howard and Rees, 1996; Seefeldt and Dean, 1997; Smith,
1994, 1999; Smith et al., 1995; Tikhonovich et al., 1995; Likhtenshtein and Therneley,
1995; Thikhonovich et al., 1995; Shilov, 1997; Therneley and Dean, 2000; Rees and
Howard, 2000; Chiu et al., 2001; Elmerich, 2001; Syrtsova and Timofeeva, 2001).

3.1.2. STRUCTURE AND PHYSICO-CHEMICAL PROPERTIES OF THE
NITROGENASE ACTIVE SITES.

The first indirect information about structure of nitrogense metal-containing active sites
was obtained in the early 1970's employing spin and electron-density labeling (Syrtsova
et al., 1971, Likhtenshtein et al. 1973, 1980), ESR and Mössbauer spectroscopy (Ohrme-
Johnson et al., 1972, 1978; Münck et al., 1975). The replacement of iron atoms in FeP
and FeMoP from Azotobacter vinelandii for para-chloromercury benzoate derivative of
nitroxide spin labels has led to spin-labeled preparations whose ESR spectra showed
strong exchange interaction similar to those for spin-labeled pea ferredoxin. The electron
micrographs of nitrogenase preparations, in which Fe atoms were substituted for
mercury atoms, revealed electron-scattering granules related to the formation of closely
arranged ensembles with four to six mercury atoms in each. These factors indicate that
FeP and FeMoP belong to the class of non-heme iron-sulphur proteins.
More detailed information about structure and spectral properties of the nitrogenase
Fe-clusters were obtained by a combination of physical methods. The structure
suggested at that t e and variation of spectra parameters is presented in Fig. 3.1, which

was plotted on the basis of the data obtained in the works of Ohrme-Johnson

im

' s and

s groups cited above. Subsequent investigations have confirmed the main
parameters and added some important details.
The principle advances in the area has been made using x-ray structural analysis.
Crystallographic data have been first produced for the nitrogenase complex of FeP (A2)
and FeMoP (A

Münck '

) from Azotobacter vinelandii (Kim and Rees, 1992) and for the
corresponding complex of Cp2 and Cp

1

1 from Clostridium pasterianum (Bolen et al.,

1993), ). A 1.6 resolution X-ray crystallographic structure of Klebsiella pneumoniae

proteins has been recently reported (Mayer et al., 1999) It was shown that FeMoco sites
in A

Å

1 , Cp 1 , and Kp 1 are 70 Å apart and FeMoco and P clusters are separated by about

19 . X-ray structures of the nitrogenase complex and the active site clusters are

presented in (Figs. 3.2-3.4).

Å