138 INSTRUMENTAL METHODS
interactions (see Sections 3.5.2 and 3.5.3 ) that are too small to be resolved
within the natural width of the EPR line. Usually, the hyperfi ne splitting values,
A , detected by ENDOR, range from 2 to 40 MHz. In transition metal com-
plexes and metalloproteins, magnetic nuclei such as^1 H,^2 H,^13 C,^14 N,^15 N,^17 O,
(^31) P, and (^33) S can be detected by ENDOR as being in the vicinity of paramagnetic
metal ions such as high - spin Fe(II) or high - or low - spin Fe(III). In the current
example the^57 Fe ENDOR spectra were obtained with protein from Azobacter
vinelandii grown on^57 Fe enriched media. 37,38 The high - fi eld g 3 and low - fi eld g 1
edges of the EPR spectrum (see previous paragraph) were used to generate
the ENDOR spectra assuming that, in different ENDOR experiments, mole-
cules were oriented with theirg 3 and g 1 axes approximately parallel to the
applied magnetic fi eld.^39 The spectra revealed that there were fi ve distinguish-
able (inequivalent) iron sites designated A^1 , A^2 , A^3 , B^1 , and B^2 for the
Azobacter vinelandii M center. It is not possible to count the total number of
irons in the spectrum using the ENDOR technique, however, subsequent X -
ray crystallography^40 indicated seven iron atoms in the M center cluster (fi ve
of which are inequivalent).
In reference 41 , the authors take the ENDOR data described above and
extend its conclusions using M ö ssbauer spectroscopy. Specifi cally,^57 Fe enrich-
ment of nitrogenase ’ s M center is used to identify all seven irons present.
Between the time of the EPR and ENDOR studies described in the previous
paragraphs and the M ö ssbauer study presented in reference 41 , the X - ray
crystallographic structure of nitrogenase was published.^40 The X - ray studies
revealed that the M center consists of two cuboidal fragments, [Mo – 3Fe – 3S]
and [4Fe – 3S], these being linked by three sulfi de bridges and attached to the
nitrogenase protein via cysteine and a histidine ligands. The M center ( S = 3/2)
is further classifi ed as being in the M N state to distinguish it from the diamag-
netic M OX ( S = 0) moiety not found in vivo and the M R EPR - silent ( S > 1 )
reduced state foundin vivo in the presence of nitrogenase ’ s Fe - protein subunit
and MgATP. The reference 41 authors identifi ed the seventh Fe site (A^4 ) as
having an unusually small and anisotropic magnetic hyperfi ne coupling con-
stant, A ≅ − 4 MHz. They also identifi ed the previously identifi ed (by ENDOR)
B^1 site as representing two equivalent Fe sites having the same hyperfi ne
interactions. The following values for the isomer shifts, δ , in mm/s are reported
in reference 41 for M N : A^1 , 0.39; A^2 , 0.48; A^3 , 0.39; A^4 , 0.41; B^1 , 0.33; and B^2 ,
0.50; this yielded a δavg = 0.41 mm/s. The δavg = 0.41 mm/s for M N differs by only
0.02 mm/s from that found for M R , whereas M N and M OX differ by 0.06 mm/s.
Because M N and M R have very similar high - fi eld M ö ssbauer spectra, the
authors believe that the iron oxidation and spin states remain the same in both
forms while the molybdenum, Mo, ion becomes reduced in the M R state. The
conclusion is supported by the fact that the magnetic hyperfi ne interactions
for M R and M N are quite similar. The δavg difference for the M N versus the M OX
pair led the researchers to conclude that the redox events (leading to
nitrogenase ’ s production of ammonia from dinitrogen) center on the iron
ions of FeMo cofactor.