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NADH-Induced Changes of the Nickel Coordination within the Active Site of the Soluble Hydrogenase from Alcaligenes eutrophus XAFS Investigations on Three States Distinguishable by EPR Spectroscopy.

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NADH-Induced Changes of the Nickel
Coordination within the Active Site of the
Soluble Hydrogenase from Alcaligenes
eutrophus: XAFS Investigations on Three States
Distinguishable by EPR Spectroscopy**
Arnd Muller, Andreas Erkens, Klaus Schneider,
Achim Muller, Hans-Friedrich Nolting,
Vicente Armando Sole, and Gerald Henkel*
Dedicated to Professor Giinter Schmid
on the occasion of his 60th birthday
4 4
-0.5
-1 5
-
-3.5
-2.5
lg c / M ( h CCI,)
-4.5
Figure 3. NMR chemical shift of the amide protons in CCI, at 300 K as a function
of the logarithm of concentration: 5 ( o ) ,7 (0).N-methylacetamide (A)
Figure 4. Superposition of the most Sable conformation of 5 with that of 4.
unit 5 by rational introduction of a trans double bond and four
methyl groups. Simple introduction of a double bond into an
w-amido-alkyl-carboxamide does not suffice to induce a 8-turn
conformation, as Gellman et a1.[’O1showed with 6.[”] Conclusion: 5 is not only isostructural with a /I11 hairpin, but also
retains the conformational flexibility that is typical for peptides.
Received: March 5 , 1997 [Z10200IE]
German version: Angew,. Chem. 1997, 109, 1805 - 1807
Keywords: conformation analysis
mimetics
- hydrogen bonds
peptido-
111 a) R Hirschmann. Angew Chem. 1991. 103, 1305-1330; Angebc. Chem. In/.
Ed. Engl. 1991. 30. 1278- 1301; b) G Muller, ihid. 1996, 108,2941 -2943 and
1996.35.2767 2769.
[2] a) J. P. Schneider, J. W. Kelly, Chem. Rev. 1995, 95, 2169-2187; b) M. Kahn,
Synl<,o 1993. 821 826.
[3] T. S. Haque. 1 C. Little, S. H. Gellman, J Am. Chem. Soc. 1994, 116, 41054106. and references therein
[4] R. W. Hoffmann, Angew. Chem 1992, 104.1147- 11 57; Angew Chem. I n / . Ed.
EngI. 1992. 31. 1124-1134.
[ 5 ] J B. Ball. R. A Hughes. P. F. Alewood. P. R. Andrews, Terruhedron 1993,49,
3467 - 3478.
[6] Macromodel 4.5. Department of Chemistry, Columbia University, New York,
NY 10027 (USA).
[7] J. C . Anderson, S. V. Ley. S . P. Marsden, Terraheriron Left. 1994, 35, 20872090.
[8] For the interpretation of IR and NMR data, see for example a) S . H. Gellman,
G . P. Dado, G.-B. Liang. B. R. Adams, J Am. Chem. Soc. 1991, 113, 11641173: b) G.-B Liang. J M. Desper, S. H. Gellman, i h d . 1993, 1l5, 925-938.
[9] R. Gijttlich. B C. Kahrs, J. Kruger, R. W. Hoffmann, Chem. Commun. 1997,
247--251.
[lo] R. R Gardner. G.-B Liang, S. H. Gellman, J Am. Chem. Soc. 1995, 1 / 7 ,
3280-3281.
[I 11 With two methyl suhstituents at the double bond of 6. a conformation corresponding to a /f turn can be induced due to 1,3-allylic strain [lo]
Angcw Cheni I n / . E d Engl. 1997. 36. No. 16
Hydrogenases are enzymes that catalyse the reversible activation of molecular hydrogen in numerous aerobic and anaerobic
microorganisms.[’] This capabiiity has attracted increasing interest especially in view of possible applications of the catalytic
principle in industrial processes or as source for “biological
hydrogen” (hydrogen technology) .[’I
Most of the hydrogenases known today are metalloenzymes
that contain nickel and iron as essential constituents (NiFe hydrogenases) in distinction to the less widespread “iron-only”
species.[’] The nickel binding site of these enzymes shows characteristic EPR signals in specific stages of the catalytic cycle
indicating an uncommon redox chemistry. Thus, the nickel center is considered the site of hydrogen activation. The NiFe hydrogenase from Desulfbvibrio gigas has recently been the focus
of special attention since the crystal structure of this enzyme
revealed the presence of a binuclear Ni/Fe center with cysteine
sulfur bridgesc3]
The soluble NAD+-reducing hydrogenase from the aerobic
H,-oxidizing bacterium Alcaligenes eutrophus H I 6 (E. C.
1.12.1.2) is a heterotetrameric enzyme. Composed of two heterodimeric proteins of different function (86 and q),
this enzyme is of higher complexity than the “typical” heterodimeric
hydrogena~es.[~l
In addition to the nickel center, the 0,-insensitive holoenzyme contains different iron -sulfur clusters (2Fe-2S,
3Fe-4S, 4Fe-4s) and a Ravine residue (FMN) as redox-active
prosthetic groups.
In this context, we were interested to learn whether and how
the coordination of nickel changes upon reductive activation of
the enzyme and, a t the Same time to tackle the question of
possible structural relationships between the nickel centers of
the hydrogenases from A. eutrophus and from D. gigas. To this
end, high-resolution X-ray absorption spectroscopy (XAFS)
was chosen as the method of choice. This technique was developed to determine the structure in the vicinity of excited atoms,
and, in contrast to diffraction methods, can also be used to
investigate noncrystalline systems. Thus, we characterized the
soluble hydrogenase from A. eutrophus by XAFS analysis in
three different states distuingishable by EPR spectroscopy.[51
Herein we report on the evaluation of the X-ray absorption
near-edge structure (XANES) and extended X-ray absorption
[*I
[“‘I
Prof. Dr. G. Henkel. Dip].-Chem. A. Muller
Fachgebiet Anorganische Chemie der Universitiit
Lotharstrasse 1, D-47048 Duisburg (Germany)
Fax: Int. code +(203)3792110
e-mail : biohenkel(u, uni-duisburg.de
Dr A. Erkens, Dr. K . Schneider. Prof. Dr. A. Muller
Fakultit fur Chemie der Universitdt Bielefeld (Germany)
Dr. H.-F. Nolting, Dr V. A. Sole
European Molecular Biology Laboratory
Outstation Hamburg (Germany)
This work was supported by the Deutsche Forschungsgememschaft (DFG),
the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie
(BMBF). and the Fonds der Chemischen Industrie.
@> WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
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1747
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fine structure (EXAFS) regions of the Ni-K edge spectra and
show for the first time that the individual states differ especially
in the number of sulfur atoms bound to nickel, and thereby also
chemically.
Starting point of the investigation is the aerobically isolated
enzyme (form I), the nickel center of which shows no EPR
signal. Treatment with N A D H leads to a reduced form (form 2),
which shows an EPR signal characteristic for the active enzyme
(“Ni-C”). By way ofcontrast, the reduction of the enzyme with
sodium dithionite produces a state (form 3), which possesses the
EPR signals associated with reduced Fe-S clusters, but-like
the oxidized form-no Ni signal.[41The normalized X-ray absorption spectra in the region of the Ni-K edge are depicted in
Figure 1 , and Table 1 summarizes the results of the XANES
analyses. Figure 2 shows the experimentally determined
EXAFS functions.
.c
-
2
1
-
2 4 6 8 10 12 14
k&’
12,
I
r/A-
Mod (p)//k4 O5
-1- 2
2 4 6 8 10 12 14
0.0
0 1 2 3 4 5 6
r/A-
k1A-l
1.5
I
1.0
12
I
1.5
81 e,
i‘o
_.-
k3x(k)/A4-4
-8
-
--1 2
2 4 6 8 10 12 14
klk’
&(E) 0.5 -
Figure 2. Experimental nickel EXAFS (k3-weighted, left) and corresponding
Fourier transform (right); top. air-oxidized; middle: NADH-reduced; bottom:
dithionite-reduced.
I
I
0.0
1
8x0
8i‘a
a3ko
EIeV
8380
I
8400
8420
Figure 1. Normalized X-ray absorptlon spectra in the region of the Ni-K edge
(solid h e : air-oxidized; dashed line: NADH-reduced, dotted h e : dithionite-reduced) .
Fdbk 1. Parameters of the normallzed X-ray absorption spectra in the range of the
Ni-K edge (XANES).
‘
8340.7
8339 6
8341.1
1 30
1.07
1.38
kk’
i
k3x(k)lk3
-$
[a] See text. [b] Energy of the edge at a normalized intensity of 0.5. [c] Maximum
edge intensity. [d] Integrated area of pre-edge peak.
Mod (p)/A4
43
--1 2
2 4 6 8 10 12 14
1748
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim. 1997
12
8
t‘o
t
k3x(k)lk3
Mod (p)h4
43
2
1
2 4 6 8 10 12 14
kk’
0.0
0 1 2 3 4 5 6
r/A
0.0
0.5 . .
0 1 2 3 4 5 6
kk’
All three investigated forms of the enzyme feature pre-edge
peaks in accordance with nickel in a distorted octahedral ligand
environment.r61 The edge structures of all three hydrogenase
spectra also indicate an octahedral coordination of nickel. Furthermore, they suggest that nickel is predominantly coordinated
by sulfur ligands after reduction with N A D H (form 2), while the
first coordination sphere of nickel is mainly composed of lighter
atoms like nitrogen o r oxygen in the other states (form 1 and
form 3 ) .
In the first step of the curve-fitting analyses based on Fourierfiltered EXAFS functions (Figure 3, r’ = 0.9-3.1 A), the ligand
spheres of nickel were modeled by combinations of oxygen and
sulfur donor functions taking into account integral occupation
numbers with coordination numbers from four to six. The results confirm the characteristic differences derived from the
XANES spectra in a convincing manner: while in forms 1 and
3 besides four oxygen atoms only two sulfur atoms contribute to
:m
Im
-u
-
-1 2
2 4 6 8 10 12 14
t‘o
0.029
0.035
0.025
bJ
Mod (p)lA4 OLi
12
8
air-oxidized
NADH-reduced
Dithionite-reduced
1.5
0.5
0
.0
.’
-
0 1 2 3 4 5 6
r/A
Figure 3 Left: Fourier-filtered nickel EXAFS (solid line) and theoretically fitted
curve (dashed line); right: corresponding Fourier transform (solid line: experiment;
dashed line: model); top: air-oxidized; middle: NADH-reduced; bottom: dithlonite-reduced.
the binding of nickel, a coordination sphere composed of four
sulfur and two oxygen atoms must be assigned to form 2.
In the further course of the structure determination, we included additional coordination shells in the curve-fitting proce-
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dures. In the case of the NADH-treated hydrogenase (form 2,
Figure 3c, d), a significant improvement of the fit is achieved by
distributing the directly bound sulfur atoms in a 3: 1 ratio over
two shells with distances of 2.20 and 2.38 8, (fit index 0.45; the
fit with solely sulfur atoms yields a fit index of 0.64). The fit is
further improved by adding a scatterer from the series of electron-rich 3d metals at a distance of 3.07 8, from Ni to the model
(fit index 0.29). This atom is most probably an iron atom (see
below). The final results of the structure refinement are listed in
Table 2."]
,
S
I
a)
I""
om0
Table 2 Rcaulta of the curve-fitting analysis for the Fourier-filtered EXAFS functions ( r ' = o 9 -3.1 A).
Enzyme Corm [a] .Y [h]
Shell
Distance
airoxidized
4
2
I
Ni ~0
Ni-S
Ni - F e
Ni-S
2.03
133
1.71
3 02
,
NI-0
Ni S
Ni-S
Ni Fe
206
2 20
2 38
307
Ni-0
Ni-S
Ni - Fe
2.05
2.39
2.88
NADHreduced
3
1
1
dithionitereduced
4
7
I
[A] [c]
2 0 2 [A*] [d] Fit index
0.014
0.016
0.01 3
0.008
0.41
0.007
0.29
0 013
0.010
0.021
0.01 1
0 029
0 020
0.51
[a] See text. [b] Coordination number. [c] Estimated error: 0.03 A
[d] Debye Waller parameter.
In contrast. the EXAFS function of the aerobically isolated
hydrogenase (form 1, Figure 3 a) shows features of considerably
higher complexity. The strong damping behavior indicates the
presence of several shells of different backscatterer atoms, which
define the relatively large peak of the Fourier transform at
about 3 8, (Figure 3 b). Their contributions to the EXAFS are
best modeled with two additional sulfur atoms at a distance of
3.02 A and one additional metal atom 2.71 8, away from the
nickel (fit index 0.41).181The qualitative improvement of the
structural model emerges unequivocally from the development
of the fit index, which amounts to 2.04 before and to 0.86 after
introduction of'the additional sulfur atoms. The results of the
final curve-fitting analysis are summarized in Table 2.17]
The EXAFS function of the dithionite-reduced hydrogenase
(form 3, Figure 3e) resembles that of the aerobically isolated
enzyme (form 1. Figure 3a). The initial two-shell model (fit index 0.89) is likewise improved by a metal atom at a distance of
2.88 A from the nickel (fit index 0.51). However, no additional
sulfur shell of physical significance comparable to that observed
in form 1 can be derived from the EXAFS data. The results of
the final structure refinement are listed in Table 2.[']
Based on the results of the curve-fitting analyses, certain fundamental statements concerning the coordination of the nickel
atom of the hydrogenase can be derived for all three enzyme
forms, which are depicted in Figure4. In all three cases, six
ligand atoms are most likely present in a distorted octahedral
arrangement around the nickel atom.
We begin the interpretation of our results with the structural
model derived for the NADH-treated enzyme (form 2, Figure 4 b). Here the nickel atom is surrounded by four sulfur ligands, of which three form slightly shorter bonds to the nickel
than the fourth. They are supposed to be the thiolate functions
of the four conserved cysteine residues of the p-subunit of the
enzyme.'"' The remaining positions of the coordination octaheAnycir. C / w m / ! I / E d EtixI. 1997. 36, No. 16
C WILEY-VCH Verlag GmbH,
Figure 4. Schematic representations of the models for the nickel center of the hydrogenase from A . rirtrophus: a) air-oxidized; b) NADH-reduced. c) dithionitereduced; the assingments of 0-donor functions shown here arc iirhitrarily chosen
(see text)
dron are occupied by lighter atoms, which might be the oxygen
atoms of bonded water molecules. The distance of 3.07 to the
second metal atom of the active site indicates that this atom
should be linked to the nickel by two bridging thiolate functions.
This metal atom is assumed to be iron, since the hydrogenase
from A . eutrophus contains-according to chemical analysisno 3d metal other than nickel and iron.[41Regarding the number
of bridging and exogenous sulfur atoms, our proposed structure
(Figure 4b) is in good agreement with the coordination of nickel
in the crystallized hydrogenase from D. gigas. This result is
especially surprising, since the crysldllographically examined
hydrogenase is an aerobically isolated and enzymatically inactive enzyme,[3] which shows-ompared
to the hydrogenase
from A. eutvophus in its NADH-activated state- -significantly
different electrochemical and spectroscopic properties.". 41
In the aerobically isolated hydrogenase (form 1 , Figure 4a) as
well as in the preparation treated with sodium dithionite (form
3, Figure 4c), only two sulfur atoms contribute to the nickel
binding. The remaining ligands are presumably nitrogen or oxygen atoms. Apart from water molecules and/or functional
amino acid residues, the amide groups of the protein chains
might be considered the source of these atoms.181In both forms
the iron is 2.71 A (form 1, Figure 4a) and 2.88 A (form 3, Figure 4c), respectively, away from the nickel. The distinct shortening of the nickel-iron distance found here in comparison with
that in form 2 (Figure 4b) strongly indicates an additional third
bridge within the heterobimetallic center of the enzyme.
The contribution of a hydrogen atom to the coordination of
nickel in all three enzyme forms cannot be completely ruled out
by EXAFS spectroscopy, since the corresponding structural
models, in which the hydrogen is modeled by an unoccupied
coordination site, are still of physical significance. However,
such a contribution meets the fit criteria worse than the binding
situation proposed by us. The same is true for an unoccupied
coordination site.
The origin of the additional cysteine sulfur atoms that contribute to the direct binding of nickel in the EPR-active state
(form 2, Figure 4 b) is of considerable interest. In the case of the
aerobically isolated hydrogenase with only two directly bonded
sulfur ligands (form 1, Figure 4a), we observe two additional
sulfur atoms at a distance of 3.02 8,from the nickel. which might
belong to a cystine residue. A possible reason for the ligand
change within the coordination sphere of nickel upon treatment
of the enzyme with NADH would be the generation of two
D-69451 Weinheim. 1997
0570-0833/97/3616-1749 S 17 50+.50 0
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additional cysteine groups by reductive cleavage of the S-S bond
within the cystine residue (form 2, Figure 4 b; see also ref. [4d]).
According to our results the reorganization of the nickel center induced by NADH does not take place upon treatment with
dithionite. This might be due to the lacking substrate properties
of dithionite, which is not compatible with the NAD+/NADHspecific binding site of the enzyme from A . eutrophus.
In summary, we have demonstrated that the active site of the
NAD-reducing hydrogenase of A . eutrophus is a heterobinuclear complex with two cysteine bridges. In this respect, there are
striking chemical similarities with the NiFe center of the hydrogenase from D.gigas. Furthermore, it was possible for the first
time to detect a substantial change of the coordination of nickel
upon reductive activation of a NiFe hydrogenase. Whether this
property, which is characteristic for A . eutrophus, is also detectable in hydrogenases of other origin requires further investigations.['61 However, it should be noted that in earlier work a
mechanism for the reductive activation of the hydrogenase from
A . eutrophus specifically triggered by NADH has been proposed, which differs significantly from that of other hydrogena~es.1~'
Experimental Section
Cells ofA. eutrophusH16 (ATCC 17699; DSM 428) were heterotrophically cuitivated, and the soluble hydrogenase (form 1) was isolated as described in reference [4].
The protein concentration was determined by the method described by Lowry, and
the metal content was analyzed by ICP-MS. For the transformation into form 2,
two independently prepared samples of the aerobically isolated hydrogenase were
treated with NADH under an argon atmosphere (final concentration 15 and 30 mM
NADH, respectively) and shock frozen after 10min. Form 3 was obtained by
analogous treatment with a fivefold excess of Na,S,O,. These enzyme preparations
were characterized by EPR spectra prior to the XAFS measurements (for experimental details see ref. [4b]).
The enzyme solutions used for the X-ray absorption spectroscopic investigations
were concentrated by ultra-filtration in Amicon Dlaflo cells (YM 100 membrane) to
a concentration of about 1 mM. Inspection of the specific activity (starting activity:
80 PM reduced NAD per minute and mg enzyme; this corresponds to the 20-fold
value of the in VIVO activity) after the concentration step indicated no damage [4]
The samples were shock frozen and kept in a helium cryostate at 20 K during the
measurements. The X-ray absorption spectra in the region of the nickel K-edge were
recorded with the EXAFS spectrometer 111,121 of EMBL (HASYLAB at DESY,
Hamburg; storage ring DORIS II,4.5 G eV, average beam current 80 mA, Si(ll1)
double-crystal monochromator, focussing toroidal mirror, absolute calibration of
the energy axis with simultaneously recorded Bragg reflections of a silicium crystal)
according to reference [lo].
The spectra were recorded by measuring the fluorescence radiation in a range from
8210 to 9100 eV with a 13-element solid-state detector (Canberra). The measuring
time was 1-2 s per point with about 1400 points per spectrum A total of 41 (form
I ) , 27 (form 2), and 28 (form 3) single scans, respectively, were averaged after
individual energy calibration. Since the individual spectra did not change during the
corresponding measuring sequences, we can exclude radiation damage. The EXAFS
functions were isolated from the averaged spectra by literature procedures, and the
determination of the background was achieved with a spline function [13]. Data
evaluation was carried out with the program packages EXPROG (energy calibration and data reduction) [14] and EXCURV88 (curve-fitting procedures employing
theoretical amplitude and phase functions) [15].
[5] a) G . Henkel. J. Kreutzberg, A. Miiller, A. Miiiler, K. Schneider, C. Hermes,
Nolting, HASYLAB Annual Report, Hamburg 1993,679; b) A. Miiller,
A. Erkens, K. Schneider, A. Miiller, H.-F. Nolting, G. Henkel, J. Inorg.
Biochem. 1995,59,643; c) A. Miiller, A. Erkens, K. Schneider, A. Miiller, H.-F.
Nolting, V. A. SolC, G. Henkel, HASYLAB Annual Report, Hamburg 1995,
vol. 11,917.
[6] a) G. J. Colpas, M. J. Maroney, C. Bagyinka, M. Kumar, W. S. Willis, S. L.
Suib, N. Baidya, P. K Mascharak, fnorg. Chem. 1991, 30, 920; b) M.
Kockerling, Dissertation, Duisburg, 1992.
[7] During the final cycles of the structure refinement, the occupation numbers
were kept at constant values, while a common value for E o , the distances of the
individual shells from the central atom, and the Debye- Waller parameters
were refined.
181 Recently diatomic molecules with triple-bond systems were postulated as additional ligands bound to nickel in the hydrogenase from Aicaligenes etrirophus
on the basis of FT-IR investigations (T. M van der Spek, A. F. Arendsen, R. P.
Happe, S. Yun, K. A. Bagley, D. J. Stufkens, W. R. Hagen, S. P. J. Albracht,
Eur. J Biochem. 1996,237,629) A diatomic molecule in a linear arrengement
(e.g. CN- or CO), which was tentatively introduced to the structural model for
that reason, did not yield any results of physical significance in the curve-fitting
analysis.
[9] A. Tran-Betcke, U. Warnecke, C. Bocker, C. Zaborosch, B. Friedrich, J Bacrerioi. 1990, 172, 2920.
[lo] G. Henkel. A Miiller, S. WeiOgraber, G. Buse, T. Soulimane, G. C. M Steffens, H:F Nolting, Angew. Chem. 1995. 107, 1615; Angew. Chem. Inr Ed.
Engl. 1995.34, 1488.
[ l l ] C. Hermes, E. Gilberg, M. H. J. Koch, Nucl. fnsrr. Mefh. 1984, 222, 207.
[12] R. F. Pettifer, C. Hermes, J. Appl. CrystaNogr. 1985, f8, 404.
[13] B. K. Teo, EXAFS: Basic Principles and Dara Ann
[la] H:E Nolting, C. Hermes, EXPROG: EMBL EXAFS Data Analysis and
Evaluation Program Package, Hamburg, 1992.
[15] N. Binsted, S. J. Gurman, J. W. Campbell, SERC Daresbury Laboratory EXCURV88 Program, Daresbury, UK, 1988.
[16] Note added in proof (received on July 14,1997): A recent XAFS investigation
(2. Gu, J. Dong, C. B. Allan, S. B. Choudhury, R. Franco, J. J. G. Moura, I.
Moura, J. LeGall, A. E. Przybyla, W. Roseboom, S. P. J. Alhracht, M. J. Axley,
R. A . Scott, M. J Maroney,J Am. C h e m Sor. 1996,/18,11lSS)shows that the
hydrogenase from A . eutruphus differs from other enzymes with respect to its
behavior described here.
A Facile Cycloisomerization for the Formation
of Medium and Large Rings via Allenes
Barry M. Trost,* Pierre-Yves Michellys, and
Vincent J. Gerusz
-
The importance of macrocycles is readily apparent by the
large number of biologically significant molecules that possess
such a core, be it a carbocycle or heterocycle.['.21 Such ring
systems may be thought to represent a compromise in the attempt to introduce conformational rigidity into acyclic structures in order to enhance biological potency. In small and normal-sized rings, the limits on flexibility mean that a ligand must
be just right for the receptor, otherwise it will not fit properly.
Medium and large rings have well-defined conformations also;
however, a number of conformations frequently will lie close in
energy to the global minimum. Thus, if the design is not perfect,
the molecule may still be able to adapt itself to fit the receptor
without an inordinate cost in energy. Such a strategy for enhancing biopotency may be more routinely used if the methods
[l] a) The Bioinorganic ChemlstryofNickel, (Ed.: J. R. Lancaster Jr.), VCH, Weinheim, 1988; b) R. Cammack, Adv. Inorg. Chem. 1988, 32, 297; c) R. P
Hausinger, Biochemistry of Nickel, Plenum, New York 1993; d) S . P. J.
Albracht, Biochim. Biophys. Acra 1994, 1188,167, and references therein.
[2] R. Cammack, Nature 1995,373, 556
[3] A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, J. C Fontecilla-Camps, Nature 1995, 373, 580.
[4] a) K. Schneider, A. Erkens, A. Miiller, Naturwissenschuften 1996, 83, 78;
b) A. Erkens, K. Schneider, A. Miiller, J Biol. fnorg. Chem. 1996, 1, 99;
c) K . Schneider, R. Cammack, H. G Schlegel, Eur. J Biochem. 1984, 142.75;
d) K. Schneider, H. G. Schlegel, Biochim. Biophys. Acto 1976, 452, 66.
[*I Prof. Dr. B. M. Trost, Dr. P.-Y. Michellys, Dr. V. J. Gerusz
Department of Chemistry
Stanford University
Stanford, CA 94305-5080 (USA)
Fax: Int code +(415)725-0259
e-mail: bmtrost(a~leland.stanford.edu
[**I We thank the National Science Foundation and the General Medical Sciences
Institute of the National Institutes of Health (NIH) for their generous support
of our programs. We also thank the Bourse Lavoisier and RhBne-Poulenc for
partial support for PYM and VJG, respectively. Mass spectra were provided by
the Mass Spectrometry Facility, University of California San Francisco, supported by the NIH Division of Research Resources.
Received: November 19, 1996 [Z9782IE]
German version: Angew. Chem. 1997, 109, 1812-1816
Keywords: enzymes
iron nickel
-
1750
- EXAFS spectroscopy
hydrogenases
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Angew. Chem. Inr. Ed Eng/ 1997, 36, No 16
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