Mössbauer Spectroscopy of Microbial Iron
Metabolization
Berthold F. Matzanke, Matthias Brandenburger,
Lars H. Böttger
Universität zu Lübeck, Isotopenlabor TNF and Institut für Physik
Ratzeburger Allee 160, D-23538 Lübeck
E-mail: matzanke@physik.uni-luebeck.de
A. Boughamoura, B.F. Matzanke, L. H. Böttger, S. Reverchon, E. Lesuisse, D. Expert, T.
Franza: Journal of Bacteriol 2007, Dec. 28 e-pub
B. F. Matzanke: Iron transport: Siderophores in: Encylopedia of Inorganic Chemistry, 2nd ed.,
R.B. King editor, Vol. 5, 2619-46, Wiley 2005
L. Rauscher, D. Expert, B.F. Matzanke, A.X.Trautwein:.J. Biol.Chem. 277, 2385-95 2002
B.F. Matzanke, G. Berner, E. Bill, A.X. Trautwein, G. Winkelmann: Biol. Metals, 4, 181-5 1991
B. F. Matzanke, E. Bill, A.X. Trautwein, G. Winkelmann: Hyperf. Interact. 58, 2359-64 1990
B.F. Matzanke, G. Müller, E. Bill, A.X. Trautwein: Eur. J. Biochem. 183, 371-9 1989
R1
R2
O
C
NH
C
O
C
R3
NH
R3
O
NH
C
O
N
C
O
O
NH
O
O
NH
C
C
N
Fe
O
O
O
N
C
NH
R3
C
O
Fig.1
Fig. 2
Iron is of central importance for many metabolic
processes: Iron is found in the active centers of
enzymes which are involved in energy (=ATP)production via electron transfer in respiratory enzymes
(aerobically and anaerobically), participates in the
primary process of bacterial photo-synthesis (charge
separation), and DNA-biosynthesis (ribonucleotide
reductase). They performe reactions with inorganic
substrates (oxygenases, dioxygenases, hydrogenases,
nitrogenases, sulfitereductases, oxygen- and NOsensors, CO2-fixation), citric acid cycle (aconitase),
Iron availability in nature: fourth most abundant
element of the earth´s crust. With the evolution of
oxygenic photosynthesis 2x109 years ago:
4 Fe2+ + O2 + 10 H2O  4 Fe(OH)3 ↓+ 8 H+
Solubility product of Fe(OH)3:
KL = 10-38.3 at pH 7: [Fe3+] = 10-17.3 M
Microrganisms need: [Fe] = 10-6 – 10-4 M. The microbial strategy is biosynthesis of iron complexing
agents (siderophores, Fig.1 ferrichrome(Fc)) which are
excreted and the ferric iron complexes of which are
taken up by highly sophisticated transport systems (Fig.
2: proteins Fhu, Iut, TonB, Exb in E.coli)
Typical Mössbauer spectrum of a siderophore
Fig. 3: Mössbauer spectrum of a frozen aqueous solution of [57Fe3+]-ferrioxamine B (12 mM)
employing BSA (100 mM) as a dilutant to minimize spin-spin relaxation. The solid line represents a
simulation based on a spin Hamiltonian: line width = 0.35 mm s–1; zero-field splitting, D = 1.2 cm–1;
rhombicity parameter, l=E/D = 0.33;  = 0.52 mm s–1; EQ = –0.84 mm s–1; asymmetry parameter,
 = 1; and isotropic hyperfine coupling tensor: Axx/gNµN = Ayy/gNµN = Azz/gNµN = –22.1 T. The
simulation does not completely fit the experimental data. This discrepancy is caused by relaxation
effects that are not dealt with in the spin Hamiltonian simulation (for corresponding theory: see next
transparency).
g
g
S and I are the electron and nuclear spin vector operators, respectively, and
their projections on to the principal axes; g, A, and gN are matrices, B is the
magnetic flux density vector, and Q, the quadrupole moment, a secondrank tensor; mB and mN are the Bohr and the nuclear magnetons,
respectively; D is essentially a scaling factor for the splitting of the
Kramers doublets. In trigonal fields the crystal field splitting is given by
DCF = 6D, separating the doublets at a ratio 4:2. E represents deviation
from axial symmetry and affects the separation of the Kramers doublets.
For D = 3E the three Kramers doublets are separated symmetrically by
3.53D which corresponds to a complete rhombic distortion (symmetries
lower than C3). The rhombicity parameter l is defined by the ratio of E/D
with 0  l 1/3. A simulation based on the formalism described in
equation (1) is shown in Fig. 3.
57Fe-uptake
in Pantoea
agglomerans
Fig. 4: Mössbauer spectra measured at 4.3 K, in a
field of 20mT perpendicular to the γ-beam:
A) 57Fe-ferrioxamine E (fox E) in frozen aqueous
solution. Cells were grown in iron deficient media to
OD578= 0.8 and then incubated with 57Fe-fox E.
B) Pantoea, 30 min incubation
C) Pantoea 60 min incubation, D) Pantoea 90 min
incubation
time
(mms-1)
EQmms-1
percentage
component 1 [Fe3+(06)]
30 min
0.47(4)
0.85(5)
13.6
60 min
0.53(4)
0.88(4)
13.3
90 min
0.49(4)
0.91(4)
29.6
OD578=1.0
0.43(2)
0.97(2)
50.1
component 2 [Fe2+(O6)]
30 min
1.28(2)
3.10(2)
86.4
60min
1.24(2)
3.06(2)
86.7
90 min
1.25(2)
3.04(2)
70.4
OD578=1.0
1.23(2)
2.86(3)
49.9
Conclusions:ferrioxamine E is
not accumulated intracellularily.
The main metabolite formed initially is an octahedral ferrous high
spin compound ([Fe2+(OX)6]. This
requires iron reduction i.e. reductase activity. The ferrous iron species was isolated and represents an
oligomeric sugar phosphate (R.
Böhnke, BF Matzanke, Biometals 8, 223-30
1995)
A second ferric ion species is
growing in, the nature of which is
not completely clear yet. Probably,
it corresponds to an [4Fe-4S]cluster (S=0).
The corresponding scheme is
shown in Fig.5
Fig.5
siderophore
siderophore
Iron metabolization kinetics in E. coli: In some strains of E. coli (endogeneous
siderophore: enterobactin) an array of major iron metabolites can be discriminated by Mössbauer
spectroscopy thus allowing a kinetics of the iron metabolite pattern. In this study an enterobactin
negative mutant (entC) was employed and iron transport occured via ferricrocin (Fc-transporter).
[4Fe-4S]
 (mm/s)
EQ (mm/s)
 (mm/s)
[Fe(II)(OX)6]
Area
Area (in %)
 (mm/s)
EQ (mm/s)
 (mm/s)
Area
Area (in %)
1,06
46,3
Low iron medium, 57Fe ferricrocin added to the inoculum MB1
0,46
1,16
0,44
Low iron medium,
0,47
1,23
0,41
0,71
57Fe
31,06
1,26
3,07
0,62
ferricrocin added to the inoculum, high pO2 MB3
0,48
20,79
1,29
3,10
0,60
1,25
54,19
Low iron medium, 57Fe ferricrocin added to the inoculum, low pO2 MB4
0,46
1,07
0,46
0,7
36,16
1,26
3,09
0,56
0,93
47,64
Low iron medium, 57Fe-ferricrocin added at OD578= 0.8, Additional growth: 30 min MB5
0,46
1,11
0,41
0,17
12,86
1,32
3,10
0,56
1,04
78,39
Low iron medium, 57Fe-ferricrocin added at OD578= 0.8, Additional growth: 180 min MB6
0,44
1,24
0,38
0,57
18,29
1,31
rubredoxin
 (mm/s)
EQ (mm/s)
 (mm/s)
3,10
0,28
0,58
1,39
44,40
ferritin (Ftn, Bfr)
Area
Low iron medium,
0,75
3,06
Area (in %)
57Fe
0,14
 (mm/s)
EQ (mm/s)
 (mm/s)
Area
Area (in %)
0,37
16,41
ferricrocin added to the inoculum MB1
6,23
0,44
0,65
0,47
Low iron medium, 57Fe ferricrocin added to the inoculum High pO2 MB3
0,74
3,11
0,39
0,14
6,24
0,49
0,67
0,42
0,43
18,77
Low iron medium, 57Fe ferricrocin added to the inoculum low pO2 MB4
0,74
3,11
0,39
Low iron medium,
0,74
3,11
0,12
57Fe-ferricrocin
0,35
0,02
6,17
0,49
0,61
0,41
0,19
10,03
added at OD578= 0.8, Additional growth: 30 min MB5
1,30
0,49
0,64
0,32
0,10
7,45
Low iron medium, 57Fe-ferricrocin added at OD578= 0.8, Additional growth: 180 min MB6
0,74
3,11
0,35
0,12
3,75
0,49
0,67
0,43
1,05
33,56
MB 1 represents the fit results derived from the steady state spectrum of entC and MB2 of
the wild-type strain. The results are similar and 4 different metabolites can be discriminated:
A putative [4Fe-4S]-cluster, [Fe(II)(OX)6], bacterial ferritin (Ftn) and a small amount of
[Fe(II)S4] (rubredoxin-like). In MB4, the total iron level is lower than under highly
oxygenated conditions (MB4), because it contains less Fe(II) and ferritin. The Fe(II)
contribution in the highly oxygenated sample MB3 is one of the highest in this study. This
is an indication for regulation patterns different from current views. Samples MB5 and
MB6 represent the kinetics of iron metabolization and are compared to the steady state
sample MB1. Again in MB5 over 80% of total iron within the short incubation time (30
min) corresponds to [Fe(II)(OX)6], indicating that reductive decomplaxation dominates the
metabolization process. After 3h (MB6), the metabolite pattern shifted dramatically. MB6
displays the highest iron accumulationin in this study, which is reflected by the largest
amount of Fe(II) and ferritin. In contrast, rubredoxin and 4Fe-4S levels are slightly lower
than under steady state conditions. This is most likely due to the still high activity of the
iron uptake apparatus and a slower cellular turnover at the stationary growth phase
(OD578=1,2). As a consequence, excessive iron is rapidly stored in ferritin in order to
circumvent oxygenic stress associated with the Fenton reaction. After 3 hours of
incubation, the metabolic pattern is obviously significantly different compared to the
metabolic state of MB1. Different iron chelates (MB7, MB8) also affect the iron metabolite
pattern, again, indicating differences in the regulatory tuning.
In summary, this study demonstrates that Mössbauer spectroscopy is a
powerful tool for deciphering distribution and regulation patterns of main
iron metabolites in microorganisms and probably also in eukaryotic cells.