Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential.. Quantification of the EPR
Trang 1The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina
Sergei Kurkin1, Jo¨rn Meuer2, Ju¨rgen Koch2, Reiner Hedderich2and Simon P J Albracht1
1
Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, the Netherlands;2Max-Planck-Institut fu¨r Terrestrische Mikrobiologie, Marburg, Germany
The purified membrane-bound [NiFe]-hydrogenase from
Methanosarcina barkeri was studied with electron
para-magnetic resonance (EPR) focusing on the properties of the
iron–sulphur clusters The EPR spectra showed signals from
three different [4Fe)4S] clusters Two of the clusters could be
reduced under 101 kPa of H2, whereas the third cluster was
only partially reduced Magnetic interaction of one of the
clusters with an unpaired electron localized on the Ni–Fe site
indicated that this was the proximal cluster as found in all
[NiFe]-hydrogenases Hence, this cluster was assigned to be
located in the EchC subunit The other two clusters could
therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe)4S] cluster Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed
Keywords: Ech; hydrogenase; iron-sulphur; pH dependence; redox properties
Hydrogenases catalyse the simplest chemical reaction in
nature: H2« 2H++ 2e– They are found in wide variety
of microorganisms Hydrogenases enable some organisms
to use H2as a source of reducing equivalents under both
aerobic and anaerobic conditions In other organisms the
enzyme is used to reduce protons to H2, thereby releasing
the reducing equivalents obtained from the anaerobic
degradation of organic substrates [1,2] On basis of the
transition-metal content, hydrogenases can be divided into
two major classes [3]: the [Fe]-hydrogenases [4] and the
hydrogenases [5–7] The large subunit of
[NiFe]-hydrogenases harbours the binuclear Ni–Fe active site,
which is coordinated by two conserved CxxC motifs, one
located in the N-terminal region and the second located in
the C-terminal region of the polypeptide [5] The small
subunit of all [NiFe]-hydrogenases displays a conserved
amino acid sequence pattern, CxxCxnGxCxxxGxmGCPP
(n¼ 61 to 106, m ¼ 24 to 61 [5]), binding one [4Fe)4S]
cluster This cluster is within 14 A˚ of the active site [8] and is
called the proximal cluster In most, but not all enzymes, the
small subunit contains six to eight additional cysteine
residues, which harbour two more clusters: in the
Desulf-ovibrio gigasenzyme these are a second [4Fe)4S] cluster
(distal cluster) and a [3Fe)4S] cluster (medial cluster) The
combination of the Ni–Fe active site and the proximal
[4Fe)4S] cluster seems to be important for the catalytic action of [NiFe]-hydrogenases [7]
The study of hydrogenases in methanogens led to the discovery of a third class of hydrogenases, not containing any metals [9] This class of enzyme is active only in the presence of its second substrate, N5,N10 -methenyltetra-hydromethanopterin There is evidence for an unknown nonmetal prosthetic group in this enzyme [10,11] Metha-nogens also contain [NiFe]-hydrogenases and the expression
of the several enzymes depends on the available energy sources [12,13] Some time ago a membrane-bound [NiFe]-hydrogenase was isolated from methanogenic archaea [14], which consists of six subunits much like hydrogenase-3 of Escherichia coli Hydrogenase-3 in E coli is part of the formate-hydrogen lyase complex and is composed of seven different subunits [15] This hydrogenase shows surprisingly little sequence homology with other [NiFe]-hydrogenases, except for the conserved residues coordinating the active site and the proximal Fe–S cluster The enzyme showed a high sequence similarity with the CO-induced hydrogenase of Rhodospirillum rubrum [16,17] The latter bacterium can grow anaerobically on CO and its [NiFe]-hydrogenase is thus expected to be insensitive towards CO The same is expected for the E coli-like hydrogenase (Ech) from Methanosarcina barkeri[14,18] From growth characteristics
of R rubrum and from cell-suspension experiments with
M barkeri, it can be inferred that the [NiFe]-hydrogenases
in these organisms probably act as a proton pumps [16,19] Ech is the only enzyme of this subclass which has been purified and partly characterized
Purified Ech consists of six subunits, encoded by genes organized in the echABCDEF operon The EchA and EchB subunits are predicted to be integral, membrane-spanning proteins, while the other four subunits are expected to extrude into the cytoplasm (Fig 1) Amino acid sequence analysis of the cytoplasmic subunits points to the presence
of two classical [4Fe)4S] clusters in EchF and one [4Fe)4S]
Correspondence to S P J Albracht, Swammerdam Institute for Life
Sciences, Biochemistry, University of Amsterdam, Plantage
Muidergracht 12, NL-1018 TV Amsterdam, the Netherlands.
Fax: + 31 20 5255124, Tel.: + 31 20 5255130,
E-mail:asiem@science.uva.nl
Abbreviations: Ech, membrane-bound hydrogenase of Methanosarcina
barkeri; EPR, electron paramagnetic resonance; Hdr,
heterodi-sulphide reductase.
(Received 8 August 2002, revised 4 October 2002,
accepted 21 October 2002)
Trang 2cluster in EchC The EchE subunit belongs to the family of
the large subunits in [NiFe]-hydrogenases and shows the
characteristic binding motif for the Ni–Fe site found in the
large subunits of all [NiFe]-hydrogenases Chemical analysis
revealed the presence of Ni, nonheme Fe and acid-labile S in
a ratio of 1 : 12.5 : 12 [18], corroborating the presence of
three Fe–S clusters A low-potential, soluble ferredoxin
(E0¢ ¼) 420 mV) was found to be the natural donor/
acceptor of electrons for Ech [18] Kinetic analyses revealed
that purified Ech is inactivated by O2and, like most
[NiFe]-hydrogenases, is inhibited by CO [18]
The biological role of Ech was recently studied using
mutational analysis [20] There are several functions
proposed for Ech, depending on the growth conditions
and cell energy requirements In acetoclastic
methanogen-esis, Ech catalyses H2formation from reduced ferredoxin,
generated by the oxidation of the carbonyl group of acetate
to CO2 Under autotrophic growth conditions, the enzyme
catalyses the energetically unfavourable reduction of
ferre-doxin by H2, most probably driven by energy-induced
reversed electron transport, and the reduced ferredoxin thus
generated functions as the low potential electron donor for
the synthesis of pyruvate in an anabolic pathway The
reduced ferredoxin also provides the reducing equivalents
for the first step of the methanogenesis, namely the
reduction of CO2to formylmethanofuran
The six subunits of Ech show a striking amino acid
sequence similarity with six subunits of proton-pumping
NADH : ubiquinone oxidoreductase (complex I) [14–16]
Complex I catalyses electron transfer from NADH to
ubiquinone and couples it to the translocation of four to five
protons across a membrane Studies of submitochondrial
particles have demonstrated that of all the Fe–S clusters of
complex I, only two, called the clusters 2 or N-2, which are
presumably located in TYKY subunit (homologous to
EchF) [21], are directly involved in energy transduction It is
known that the redox potential of these Fe–S clusters is pH
dependent ()60 mVÆpH unit)1) [22], which is rare for Fe–S
clusters The TYKY and EchF subunits belong to a family
of polypeptides, which are found exclusively in complex I and proton-pumping hydrogenases [23] The amino acid sequences of the proteins in this family are so unique and conserved, that the two [4Fe)4S] clusters held by this protein were proposed to function as the direct electrical driving unit for a proton pump [23] To delineate a possible role of the Fe–S clusters in the Ech of M barkeri in this action, the electron paramagnetic resonance (EPR) and redox properties of these Fe–S clusters were investigated
M A T E R I A L S A N D M E T H O D S
Purification ofM barkeri Ech and sample preparation Ech was purified as described elsewhere [18] The enzyme was routinely dissolved in 50 mM Mops pH 7.0, 2 mM
dithiothreitol and 2 mMdodecylmaltoside under an atmo-sphere of 4% (v/v) H2 For redox titrations the concentra-tion of dithiothreitol in the enzyme soluconcentra-tion was reduced to
2 lM The following buffers were used for redox titrations:
100 mM Tris/HCl pH 8.0, potassium phosphate pH 7.0, Tris/Mes pH 6.5, or Mes pH 6.0 The standard enzyme solution was concentrated and then diluted with new buffer; this was repeated several times Samples for all spectro-scopic measurements were handled anaerobically i.e all operations were performed in anaerobic box at 4% (v/v) H2 Membranes were obtained from cells grown on acetate at
37C and were prepared as described [18] They were suspended in 50 mM Mops/NaOH pH 7.0, containing
2 mM dithiothreitol Ferredoxin was purified as described
by Fischer and Thauer [24]
Redox titrations Redox titrations of Ech were performed using a Pt vs Calomel electrode system (Radiometer, Copenhagen) in a device analogous to that of Dutton [25] The redox potential was measured using a digital voltmeter RW9408 (Philips) All redox potentials mentioned here are expressed vs the normal hydrogen electrode Correction for the temperature dependence of the reference electrode was performed as in Ives and Janz [26] As Ech is rapidly inactivated by O2, several precautions were taken to avoid the introduction of
O2 into the titration cell First, the cell was flushed with 100% (v/v) H2(freed from traces of O2by passing through a column with a Pd catalyst; Degussa, type E236P) There-after, a solution of Ech (incubated under 100% H2) was transferred anaerobically into the titration cell Two types of titrations were performed, one in the presence of redox dyes and one in the absence of these dyes In both cases the cell was continuously flushed with a water-saturated mixture of
H2and He, used to adjust the redox potential in the system The home-built H2/He mixer produced mixtures from 0.1%
to 100% (v/v) H2[27] In this system the potential values read from the Pt electrode were within 10 mV of the theoretical redox potentials calculated from the gas mixture using the formula:
Eh¼ RT
Flog epH RT
2F log elog PH2
where R is the gas constant, F is the Faraday constant and T
is the temperature in Kelvin
Fig 1 Schematic representation of the possible organization of the
subunits of Ech in membranes from M barkeri The Ni–Fe active site in
the EchE subunit together with the proximal cluster located in EchC
subunit form the centre for hydrogen production Two transmembrane
proteins EchA and EchB are supposed to be involved in the transfer of
protons across the membrane The two [4Fe )4S] clusters in subunit
EchF, which is related to the TYKY subunit in bovine complex I, have
been suggested to be involved in proton translocation coupled to
electron transfer [23].
Trang 3In redox titrations in the presence of mediating dyes the
following dyes were present in a final concentration of
50 lM: 2,3,5,6-tetramethyl-p-phenylendiamine
dihydrochlo-ride (E0¢ ¼ +275 mV), 2,6-dichlorophenol-indophenol
(E0¢ ¼ +230 mV), 1,2-naphtoquinone-4-sulfonic acid
(E0¢ ¼ +215 mV), phenazine methosulfate (E0¢ ¼
+80 mV), 1,4-naphtoquinone (E0¢ ¼ +36 mV), methylene
blue (E0¢ ¼ +11 mV), duroquinone (E0¢(1,2) ¼) 5/
+35 mV), indigodisulfonate (indigo carmine; E0¢ ¼
)125 mV), 2-hydroxy-1,4-naphtoquinone (E0¢(1,2) ¼
)139/)152 (mV), lapachol (E0¢ ¼)179 mV),
antraqui-none-2-sulfonate (E0¢ ¼)225 mV), safranin T (E0¢ ¼
)289 mV), benzyl viologen (E0¢ ¼)358 mV) and methyl
viologen (E0¢ ¼)449 mV) All redox potentials are given at
pH 7 As some of the redox dyes have a pH-dependent
redox potential, these values are not valid for the titrations
performed at pH 6 or pH 8 However the mixture of
these dyes still covers the whole redox-potential range at pH
6 or pH 8 Also in this case the redox potentials were set by
a H2/He gas mixture As this limits the potential range,
potentials higher than that of 0.1% (v/v) H2were achieved
by addition of aliquots of potassium ferricyanide (250 mM)
as oxidizing agent or, to bring the potential down again,
by aliquots of a solution of sodium dithionite (100 mM)
as reducing agent After stabilization of the redox potential,
samples were withdrawn with a gas-tight syringe through
a suba-seal rubber stopper and injected into EPR tubes
The tubes, sealed with latex tubing, were preflushed with the
same gas (mixture) of the titration cell After filling, the
tubes were rapidly frozen by immersion in cold isopentane
(133 K)
EPR measurements
EPR spectra at X-band (9 GHz) were obtained with a
Bruker ECS 106 EPR spectrometer with a
field-modula-tion frequency of 100 kHz Cooling of the sample was
attained with an Oxford Instruments ESR 900 cryostat
with a ITC4 temperature controller The
sample-tempera-ture indication from this instrument was correct from
4.2 K to 100 K within ± 2% as ascertained from the
Curie dependence of a copper standard (10 mMCuSO4
Æ5-H2O, 2M NaClO4, 10 mMHCl) The magnetic field was
calibrated with an AEG Magnetic Field Meter The
X-band frequency was measured with a HP5350B
microwave frequency counter The microwave power
incident to the cavity was measured with a HP432B
power meter and was 260 mW at 0 dB Simulations were
performed as described [28] Quantification of EPR signals
was carried out by direct double integration of the
experimental spectra [29,30] or by comparison with a
good-fitting simulation
Analysis of titration data
The midpoint potentials of the Fe–S clusters were estimated
using the amplitudes in the EPR spectra at two different
g-values: for one signal (here termed the g¼ 1.92 signal)
the peak at g¼ 1.947 (see Fig 3, trace A) was used; for a
second signal (g¼ 1.89 signal) the amplitude of the trough
at g¼ 1.88 was taken The amplitudes were plotted against
the applied potential and each data set was then fitted to the
Nernst equation:
Eh¼ E0
0þ ð59=nÞ: log [ox]=[red]
where E0¢ is the midpoint potential in mV at the pH used, Eh
is the applied potential, n is the number of electrons involved
in the redox reaction IGOR PRO software (WaveMetrics, Inc.) was used for the curve-fitting analysis Quantification
of the EPR signal to obtain the total concentration of Fe–S clusters was performed with the samples obtained under 100% H2at pH 8 in the absence of redox mediators The Ni content of the enzyme, determined by Atomic Absorption Spectroscopy, was used as the basis for the enzyme concentration
Metal content determination Nickel was determined with an Hitachi 180-80 polarized Zeeman Atomic Absorption spectrophotometer using either internal standards or a standard series The enzyme concentrations, calculated on basis of a protein determin-ation with the Bradford method assuming molecular mass
of 180 kDa, correlated well with the values based on the Ni contents
R E S U L T S
EPR properties of Fe–S clusters in Ech EPR spectra of purified Ech A sample of the purified enzyme equilibrated with 4% (v/v) H2, either in Mes buffer at pH 6.0, or in Mops buffer at pH 7.0, showed signals only in the g¼ 2.3 to g ¼ 1.8 region, apart from a small g¼ 4.3 signal due to high-spin 3d5metal ions in a rhombic ligand field (usually adventitious Fe3+) From the temperature dependence of the signals in the g¼ 2 region for fully reduced enzyme under 100% H2 (Fig 2, left panel), it is concluded that the spectrum is due to at least two, possibly three, different signals of reduced [4Fe)4S] clusters All signals broadened considerably above 17 K Below 30 K one signal was optimally sharpened at 17 K It has a trough around g¼ 1.921 and is termed here as the g¼ 1.92 signal Its gzvalue is
at 2.050 The second major signal only sharpened optimally at 12 K and has a trough at g¼ 1.887 (termed the g¼ 1.89 signal) Its gzvalue is at 2.078 At 17 K and lower, there was also a clear shoulder (peak) detectable around g¼ 1.959 As in redox titrations (see below) this signal behaved independently of the other two signals, it is termed the g¼ 1.96 signal At this point it is unclear where the gzand gxlines of this signal are No additional signals were observed down to 4.2 K At 70 K a minute signal could be observed (at a larger magnification) with a major line around g¼ 2.3, which is reminiscent of Co2+
in methyltransferase [31] This signal was also observed in membranes of M barkeri (see below) At pH 7.0 the Fe–S signals had about twice the intensity of that found at
pH 6.0; the overall line shape of the spectrum was the same at both pH values Direct double integration of the Fe–S signals at 12 K at pH 7 amounted to a total spin concentration of 51 lM; the enzyme concentration was
25 lM As the amino-acid sequence of Ech points to the presence of three [4Fe)4S] clusters, the sample was apparently only partially reduced under the conditions used (100% (v/v) H at pH 7.0)
Trang 4To a first approximation, the spectrum of Ech under 4%
(v/v) H2 at pH 7.0 could be simulated rather well on the
basis of the two main components mentioned above (Fig 2,
right panel) Using the simulated spectra, it could be
calculated that the relative spin concentrations of the
g¼ 1.89 signal was 1.6 times that of the g ¼ 1.92 signal
In addition, a rather isotropic signal at g¼ 2.03 was
apparent, especially at higher temperatures, where the other
signals broadened The line shape and the temperature
dependence of the signal indicate a free radical Its g-value,
however, indicates that the radical cannot be a truly free
electron (with a g-value close to the free-electron value) We
also noticed that this signal could not be saturated at 4.2 K
and full microwave power This suggests that it might be
due to a radical close to a very rapidly relaxing paramagnet,
e.g high-spin Fe2+
Another method to obtain a rough impression of the line
shape of two overlapping signals with different relaxation
rates is the one described by Hagen and Albracht [32] By
setting the observing amplifier around 90 out of phase,
while partly saturating the signals with a suitable microwave
power at a suitable temperature, first one and then, at a
slightly different phase, the other signal could be virtually
eliminated from the spectrum This is demonstrated in
Fig 3 The g¼ 1.92 signal (trace B) shows an apparent gz
at 2.05 The perpendicular region (gxybetween 1.90 and
1.97) shows more structure than assumed in the simulation
of Fig 2 Also the peak at g¼ 1.96 is clearly detectable, as well as a gz-like peak at 2.01 We tentatively conclude that the spectrum represents an overlap of two different signals, i.e the g¼ 1.92 signal and the g ¼ 1.96 signal The
g¼ 1.89 signal (trace C) apparently has its gzvalue around 2.07 (top), while gxy line has a trough at g¼ 1.89 This agrees with the interpretation shown in Fig 2 The radical-like signal at g¼ 2.03 is present in trace C, but not in trace
B This indicates that the species causing it has a relaxation rate at 10 K which is of the same order of magnitude as that
of the g¼ 1.92 species We also note that the g-values and the temperature dependence of the g¼ 1.92 signal are similar to those of the clusters N-2 in bovine-heart complex I [21,33] As the g¼ 1.89 signal appears to interact with the observed Nia–L* signal (see below), it is concluded that this signal is presumably due to the proximal cluster in the EchC subunit and so the remaining Fe–S signals present
in the spectrum at 17 K are ascribed to the [4Fe)4S] clusters
in the EchF subunit
Two of the subunits of Ech, EchE and EchC, bear a large resemblance to the large and the small subunits, respect-ively, of [NiFe]-hydrogenases, suggesting the presence of a
Fig 2 Temperature dependence of the Fe–S signals of purified Ech
reduced with 10 1 kPa H 2 (left panel) and a simulation of the 12 K EPR
spectrum (right panel) Spectra were recorded at nonsaturating
microwave powers and replotted normalized for microwave frequency,
microwave power, temperature and receiver gain; hence they can be
quantitatively compared EPR conditions: microwave frequency,
9416.2 MHz; microwave powers incident to the cavity, 50, 40,
30, 30, 30, 20, 20 dB for spectra from top to bottom (0 dB ¼
260 mW); modulation amplitude, 1.27 mT; the temperature is
indi-cated for each spectrum In the right panel the following spectra are
presented: (A) Experimental spectrum of Ech dissolved in Mops buffer
pH 7.0 under 4% H 2 and further reduced with a few grains of solid
dithionite EPR conditions: microwave frequency, 9415.8 MHz;
microwave power, 30 dB; modulation amplitude, 0.64 mT;
tempera-ture, 12 K (B) Simulation of the g ¼ 1.89 signal with parameters
g xyz ¼ 1.88391, 1.90223, 2.06977 and widths (xyz) ¼ 5.2, 3.7, 6.0 mT.
(C) Difference spectrum A minus B This difference spectrum was used
to fit the remaining signal (g ¼ 1.92 signal) (D) Simulation of the
g ¼ 1.92 signal (trace C) with parameters g xyz ¼ 1.91821, 1.93799,
2.04721 and width (xyz) ¼ 2.77, 2.70, 2.66 mT (E) Difference
spec-trum C minus D.
Fig 3 Three EPR signals that can be detected in the spectrum of purified Ech reduced with 10 1 kPa H 2 at pH 8.0by varying the detecting phase of the amplifier (A) Normal EPR spectrum (B) Approximate line shape of the g ¼ 1.92 plus g ¼ 1.96 signal obtained by using an amplifier phase to minimize the g ¼ 1.89 signal (C) Approximate line shape of the g ¼ 1.89 signal obtained by using an amplifier phase to minimize the g ¼ 1.92 signal EPR conditions: microwave frequency, 9426.6 MHz; microwave power, 10 dB; modulation amplitude, 1.27 mT; temperature, 10 K.
Trang 5Ni–Fe active site Hence, under 4% (v/v) H2an EPR signal
due to the Nia–C* state (usually with gxyz¼ 2.20, 2.15, 2.01)
is expected, as apparent in many other [NiFe]-hydrogenases
under that H2-partial pressure No such signal was
observed, however (data not shown) Even minute signals
due to Nia–C* can usually be detected in a background of
large overlapping signals, by making use of its light
sensitivity [34] Hence a sample was illuminated for
25 min at 45 K Curiously, a difference spectrum of light
minus dark showed only the induction of a signal typical for
the Nia–L* state (gxyz¼ 2.0486, 2.101, 2.270), but no
disappearance of its expected parent Nia–C* signal could be
detected (Fig 4) The signal, which could be readily
simulated, amounted to a concentration of only 1.1 lM; it
could be clearly seen in the spectrum, however, due to its
sharp lines When studied below 15 K, a clear two-fold
splitting of the gyand gxlines, but not of the gzline, was
apparent (Ax¼ 3.9 mT, Ay¼ 5.2 mT) The splitting was
blurred at 15 K and was not apparent at 20 K or higher
temperatures (Fig 4) This temperature dependence
paral-lels the temperature dependence of the g¼ 1.89 signal In [NiFe]-hydrogenases the Nia–L* EPR signal shows a small splitting due to interaction of the Ni-based unpaired electron with the S¼ 1/2 system of the reduced proximal [4Fe)4S] cluster signal [35–37] It is therefore tentatively concluded that the g¼ 1.89 signal is due to the reduced proximal cluster in Ech Usually, in [NiFe]-hydrogenases containing a [3Fe)4S] cluster, the relaxation of the proximal cluster is very much enhanced by coupling to the nearby
S¼ 2 system of the reduced [3Fe)4S] cluster As a result the effective relaxation of the proximal cluster cools down only
at temperatures below 7 K, and it is only then when the
Nia–C* and Nia–L* signals in these enzymes show the twofold splitting Ech does not contain a 3Fe cluster and therefore the proximal cluster has relaxation properties normally associated with [4Fe)4S] clusters
Oxidation of the sample by stirring with air for a few minutes resulted in the complete disappearance of the signals due to Co2+and the reduced Fe–S clusters Only traces of lines reminiscent of the EPR signals of the Nir* state (observed here around gx¼ 2.31 and gy¼ 2.178) and the Niu* state (observed here at gx¼ 2.28 and gy¼ 2.24) appeared (data not shown) There was only a trace of a signal reminiscent of that of an oxidized [3Fe)4S] cluster and it is concluded that Ech does not contain a [3Fe)4S] cluster
EPR spectra of membranes of M barkeri As Ech (a membrane-bound protein) constitutes up to 3% of the total cell protein, we have also inspected membranes of M bark-eriwith EPR Initial EPR measurements failed due to the presence of large signals from Mn2+, which is added to the growth medium Omission of Mn2+from the medium did not result in any noticeable changes in the growth or specific activity of Ech in the membranes, and now only trace signals due to Mn2+remained Wide-scan spectra (600 mT)
at 12 K of reduced membranes thus obtained revealed only three main lines (Fig 5) The line around g¼ 2.3 is due to the gxylines of the Co2+, presumably from a membrane-bound methyltransferase The other two lines (around
g¼ 2.05 and g ¼ 1.9) are due to reduced [4Fe)4S] clusters
As signals from [4Fe)4S] clusters usually disappear at 45 K due to relaxation broadening, whereas the Co2+signal does not (Fig 5, trace B), a difference spectrum (Fig 5, trace C) reveals the spectrum of these clusters only This is shown in more detail in the right panel of Fig 5 A difference of the spectra at 12 K and 45 K shows similarities to the spectrum
of the purified Ech in Fig 5, right panel As with the purified enzyme, no additional signals could be observed in the membranes between 12 K and 4.2 K
Membranes of M barkeri contain additional metallo-proteins, like heterodisulphide reductase (Hdr), with Fe–S clusters showing EPR signals in the same region [38] and a b-type cytochrome [39], methanophenazin-reducing (F420 -nonreducing) [NiFe]-hydrogenases (VhoGAC and Vht-GAC) [40], as well as a methyltransferease The Vho, but not the Vht hydrogenase, is present in high amounts in acetate-grown cells Upon cell lysis, however, Vho hydro-genase loses its contact to the b-type cytochrome, which anchors this enzyme in the membrane The amount of the other hydrogenases (which only reduce dyes but not ferredoxin) was estimated by activity measurements Based
on these determinations it can be concluded that the amount
Fig 4 Temperature dependence of the splitting of the Ni a –L* signal An
EPR tube with Ech (in Mops buffer pH 7.0 under 4% H 2 ) was frozen
in liquid nitrogen and then kept in the dark at 200 K for 10 min A
spectrum was recorded at the indicated temperatures and then the
sample was illuminated for 25 min in the EPR cavity at 45 K [34].
After switching off the light, a second set of spectra was recorded under
identical conditions The difference spectra light minus dark are plotted
in the figure EPR conditions: microwave frequency, 9415.5 MHz;
microwave power, 20 dB (70 K, 45 K), 30 dB (20 K, 15 K, 12 K,
10 K) or 10 dB for the bottom spectrum; modulation amplitude,
1.27 mT Spectra A–F were normalized for temperature, microwave
power and gain Vertical dashed lines indicate the positions of the g y
and g lines.
Trang 6of Vho and Vht hydrogenases in washed membranes is very
low
When membranes were oxidized with air, the signals of
Co2+ and of the reduced Fe–S clusters disappeared and
now two main signals dominated the spectrum (data not
shown) They were recognized as an Niu* signal, typical for
[NiFe]-hydrogenases in the oxidized, unready state, and a
peculiar signal earlier encountered by us in purified,
oxidized preparations of Hdr in the presence of H-S-CoM
(gxyz¼ 2.013, 1.991, 1.938) from M barkeri and
Metha-nothermobacter marburgensis [38] Both signals could be
readily simulated, showing that the remainder of the
spectrum consisted of small signals presumably due to an
oxidized, low-spin cytochrome (with gz¼ 2.32) and some
contaminating Mn2+ No trace of a signal due to the Nir*
state (with gz¼ 2.31 and gy¼ 2.16) could be spotted The
simulations enabled quantification of the signals The spin
concentration represented by the Niu* signal amounted to
11.6 lM; the Hdr signal was calculated to represent a spin
concentration of 16.3 lM This indicates that the EPR
signals of the reduced Fe–S clusters from Hdr, which have
similar g-values [38], heavily interfere with those of Ech in
the used membranes and hence these membranes are not
suited for the study of the Fe–S clusters of Ech As the
membranes contain only very low amounts of other
hydrogenases, the Niu* signal is considered to be due to
Ech only; its concentration was at least 2.1 g pure enzyme
proteinÆL)1 Estimating the total protein concentration to be
80 gÆL)1, this is about 2.7% of the total membrane
protein
Redox titration in the absence of redox dyes at pH 6.5,
7 and 8 Because in some cases redox dyes have been shown to change the redox properties of [NiFe]-hydrogenases we performed redox titrations in the absence of redox dyes These titrations were performed at three different pH values (6.5, 7 and 8) using pure Ech preparations at 25C As an example of the spectral changes we have compiled spectra obtained at pH 8 and pH 7 (Fig 6) The enzyme was first incubated in the titration cell under 100% H2 At all pH values the lines at g¼ 2.05, 1.92, 2.065 and 1.89 were decreasing in amplitude with increasing potential The ratio between the g¼ 1.92 signal and the g ¼ 1.89 signal was different at different pH values The g¼ 1.89 signal was more pronounced at pH 6.5 and 7 At pH 8 a small apparent shift or the line at gz¼ 2.05 to smaller values was noticed when the potential was increasing The amplitude changes of the g¼ 1.96 line were accompanied by the changes of a kink in the 1.92 line suggesting a contribution
of the g¼ 1.96 signal at that position, as also suggested by trace B in Fig 3 The peak at g¼ 2.01 was present in titrations at all three pH values and disappeared with increasing potential At pH 8 it was no longer detectable at
H2concentrations < 10% This signal was less pronounced
at pH 7 and 6.5
The spin concentrations, estimated for enzyme under 100% (v/v) H2, were 45, 65 and 56 lMat pH 6.5, 7 and 8, respectively As the enzyme concentration used for all titrations was 25 lM, the amount of spins per enzyme molecule represented by the Fe–S signals was 1.8, 2.6 and 2.24 for pH 6.5, 7 and 8, respectively
At all three pH values a plot of the amplitude of the
g¼ 1.92 and g ¼ 1.89 signals fitted best to n ¼ 2 Nernst curves (Fig 7) The amplitudes at pH 6.5 were smaller and the data were rather scattered; hence the estimated E0¢ values are less reliable, while the n-values could not be
Fig 5 Wide-scan EPR spectra of membranes of M barkeri (left panel)
and details of the g = 2 region (right panel) Membranes from
acetate-grown cells were prepared as described in Materials and methods and
equilibrated with 100% H 2 before freezing in liquid nitrogen Spectra
were recorded between 5 and 605 mT (left panel) and a scan range of
only 80 mT was used in the g ¼ 2 region (right panel) Left panel: (A)
spectrum at 12 K; (B) spectrum at 45 K; spectra were normalized for
microwave frequency, microwave power, temperature and receiver
gain; (C) difference A minus B Right panel: (A) spectrum at 12 K; (B)
spectrum at 45 K; spectra were normalized for microwave frequency,
microwave power, temperature and receiver gain; (C) difference A
minus B (D) Spectrum of the purified enzyme at pH 7.0 under 4%
(v/v) H 2 EPR conditions: microwave frequency, 9416.5 MHz;
microwave power, 40 dB for A and D, 30 dB for B; modulation
am-plitude, 1.27 mT (left panel) and 0.64 mT (right panel); temperatures
are indicated in the figure.
Fig 6 EPR spectra of samples from a titration of Ech hydrogenase at
pH 8 (left panel) and at pH 7 (right panel) with H 2 /He mixtures (in the absence of redox dyes) EPR conditions: microwave frequency,
9460 MHz; microwave power, 30 dB; modulation amplitude, 1.27 mT; temperature, 12 K All spectra are normalized for the gain,the tube-calibration factor, and the microwave frequency and hence they can be directly compared The redox potentials are indi-cated in the figure.
Trang 7determined The midpoint potentials of the two signals
obtained at all three pH values are summarized in Table 1
For both signals there was a pH dependence of)38 to
–50 mVÆpH unit)1
Redox titrations in the presence of redox dyes at pH 6,
6.5, 7 and 8
EPR spectra under 101 kPa H2at different pH values in
the presence of redox dyes The titrations at all four pH
values were started with 100% H2-reduced enzyme, which
was transferred anaerobically to the titration vessel under a
continuous flow of O2-free H2 Hence, the starting redox
potential was that of the hydrogen potential at each pH
value Spectra taken under these conditions are summarized
in Fig 8, left panel Comparison of the four spectra shows
that the degree of reduction diminished with decreasing pH
The spin concentrations obtained by direct double
integra-tion showed that at pH 6 the intensity was only 30% of
that at pH 8 There were also clear changes in the overall
line shapes of the spectra At pH 6 two separate gzlines at
2.078 and 2.050 were observed (Fig 8, left panel) At
pH 6.5, the 2.05 line markedly increases together with the
trough around g¼ 1.92 This reinforces the earlier
inter-pretation that these two lines form the gzand the gxyregion
of the g¼ 1.92 signal, whereas the gz¼ 2.078 and the trough at g¼ 1.887 form the gz and gxy lines of the
g¼ 1.89 signal At pH 7.0 both of the two gzlines as well as the two gxy lines increased noticeably Also the g¼ 1.96 signal could now be discerned as a shoulder At pH 8.0 this shoulder at g¼ 1.959 is much better defined and forms a separate peak As the region between the two gzlines at 2.078 and 2.050 seems to fill up, one might conclude that this is caused by a gzline (around g¼ 2.06) of the g ¼ 1.96 signal Spectra encountered during the redox titrations (see below) made this interpretation less likely At this point we tentatively conclude that the g¼ 1.96 species has its gzline either at 2.06 or at 2.01
Comparison of the EPR spectra at different pH values at )340 mV It is interesting to compare the EPR spectra at different pH values and the same potential ()340 mV) The comparison showed that the overall reduction level was roughly the same (Fig 8, right panel) although there were clear spectral differences At none of the pH values was any trace of the g¼ 1.96 signal observed The relative ratio of the other two signals was clearly dependent on the pH At
pH 6.0 the g¼ 1.89 signal dominated the spectrum, while
at pH 8.0 the g¼ 1.92 signal was the most pronounced The g¼ 1.96 species apparently has a redox potential considerably lower than those of the g¼ 1.92 and g ¼ 1.89 species (see below)
Redox titrations results The overall behaviour of the 2.05/ 1.92 lines of the g¼ 1.92 signal and the 2.065/1.89 lines of the g¼ 1.89 signal in the titrations in the presence of redox dyes was comparable to the titration in the absence of these dyes (Fig 9) The g¼ 2.01 signal found in the absence
of dyes was not detectable in the EPR spectra in the presence of dyes as it was obscured by the strong radical signals round g¼ 2.00, originating from the redox dyes The spin concentrations estimated by double integration of the experimental EPR spectra of enzyme under 101 kPa H2 were 0.51, 1.8, 2 and 1.9 spins per molecule at pH 6, 6.5, 7 and 8, respectively These values are slightly overestimated due to the contribution of the radical signals The amplitudes of the g¼ 1.92 and g ¼ 1.89 signals changed with pH; they were smaller at lower pH values (see Table 1) This reflects the overall decrease in the level of reduction of the enzyme at lower pH values The line at g¼ 1.96 disappeared on shifting from pH 8 to pH 6, in line with the
Fig 7 Redox behaviour of the g = 1.92 and g = 1.89 signals in a
titration in the absence of mediating dyes at pH 6.5, 7 and 8 The
amplitudes (arbitrary units) of the g ¼ 1.92 signal (left panel) and the
g ¼ 1.89 signal (right panel) are plotted against redox potential Solid
curves indicate theoretical Nernst lines with n ¼ 2 The estimated E 0 ¢
and n-values and the maximal amplitudes of the signals are listed in
Table 1.
Table 1 Summary of the redox properties of the Fe–S clusters in Ech as obtained from the redox titrations with H 2 /He mixtures in the presence and in the absence of the redox dyes.
g ¼ 1.92 signal g ¼ 1.89 signal
pH Dyes n-value Amplitude under 1 bar H 2 E 0 ¢ (mV) n-value Amplitude under 1 bar H 2 E 0 ¢ (mV)
a Arbitrary units.
Trang 8conclusions from the EPR spectra at)340 mV at different
pH values (Fig 8, right panel) Fig 9 shows that the
g¼ 1.96 signal appeared only at the lowest potentials Its
E0¢ value is estimated to be well below)420 mV
In all titrations, but especially in those at pH 6.0 and 6.5, weak signals due to Ni were observed at H2-partial pressures
of £ 10% The signals had the characteristic g-values of the
Nia–C* state (gxyz¼ 2.21, 2.13, 2.01) and the light-induced
Nia–L* state (gxyz¼ 2.05, 2.11, 2.3), as observed in other [NiFe]-hydrogenases [34] The total spin concentration amounted to maximally 10% of the enzyme concentration The data obtained in the presence of dyes (Fig 10) were not as clear-cut as those obtained in the absence of dyes At all pH values, except pH 8, the g¼ 1.92 and 1.89 signals both titrated as n¼ 2 systems At pH 8 the best fit was obtained with n¼ 1 and this result is different from the titration in the absence of dyes, where the best fit was obtained with n¼ 2 Nernst curves
D I S C U S S I O N
Iron-sulphur clusters The best way to study membrane-bound enzymes, especially for those expected to pump protons, is to use intact membranes As demonstrated, 3% of the protein content
of membrane preparations of M barkeri consisted of Ech, but the concentration of Hdr was also quite high This prevented a specific study of the Fe–S clusters in Ech We therefore turned to the purified enzyme
From the EPR line shape and the temperature depend-ence of spectra from H2-reduced Ech, it can be concluded that signals due to three different S¼ 1/2 species from reduced [4Fe)4S] clusters are present We have labelled them as the g¼ 1.92 signal, the g ¼ 1.89 signal and the
g¼ 1.96 signal Only insignificant signals due to a [3Fe)4S]+cluster could be detected in air-oxidized enzyme This result is in line with the presence of two four-Cys motifs for the binding of [4Fe)4S] clusters in the amino acid sequences of the EchF subunit and one such motif in the EchC subunit It also is in good agreement with the content
of Fe and acid-labile sulphur of the purified enzyme The redox titrations indicated that the g¼ 1.96 signal has the lowest redox potential (well below )420 mV at pH 7); therefore this cluster could only partly be reduced This is in line with the maximal amount of spins detected in the spectra of the reduced Fe–S clusters ( 2–2.6 spins per enzyme molecule at pH 8)
The temperature dependence of the splitting of the Nia– L* signal paralleled the temperature dependence of the
g¼ 1.89 signal We hence conclude that the unpaired spin located at the Ni site has magnetic interaction with the Fe–S cluster responsible for the g¼ 1.89 signal This indicates that this [4Fe)4S] cluster is the proximal cluster located in the EchC subunit It then follows that the two [4Fe)4S] clusters causing the g¼ 1.92 and g ¼ 1.96 signals are located in the EchF subunit
A major disadvantage of the use of redox mediators in redox titrations is that they sometimes dramatically change the redox properties of [NiFe]-hydrogenases [27,41] The interaction of H2with hydrogenases offers the possibility to study redox changes in enzyme in the absence of redox mediators simply by varying the H2-partial pressure in a known mixture of H2and He This method minimizes the possible artefacts introduced by redox dyes This laboratory has used the method before for the hydrogenases from
M marburgensis and Allochromatium vinosum It was
Fig 8 EPR spectra of Ech under 101 kPa H 2 in the presence of
me-diating dyes at different pH values (left panel) and EPR spectra of Ech
from titrations poised at )340mV ± 5 mV (right panel) The measured
potential at each pH value is given in the figure and this legend The
theoretical potential of 101 kPa H 2 is given in this legend in
paren-theses Left panel: (A) pH 8, )463 mV ()472 mV); (B) pH 7,
)405 mV ()413 mV); (C) pH 6.5, )383 mV ()383 mV); (D) pH 6,
)360 mV ()360 mV) Right panel: (A) pH 8; (B) pH 7; (C) pH 6.5
and (D) pH 6 The EPR conditions were the same as in Fig 6.
Fig 9 EPR spectra (Fe–S region) of Ech during redox titrations at
potentials below )282 mV at pH 8 in the presence of redox dyes EPR
conditions were as in Fig 6 All spectra are normalized for gain, tube
factor and microwave frequency.
Trang 9observed that the presence of dyes had a major effect on the
reversible redox transition between the Nia–C* and the Nia–
SR states The reaction was an n¼ 1 transition involving
one proton when performed with a H2/He mixture in the
presence of redox dyes [27,41] When the dyes were omitted,
however, the reaction was found to be n¼ 2 and involved
two protons In addition, in the absence of redox dyes, there
was no redox equilibrium between the Nia–S and Nia–C*
states A limitation of the titrations in the absence of dyes is
the limited potential range, which can be covered by the
2H+/H2 couple The maximal obtainable potential is
approximately 120 mV above that of the hydrogen
poten-tial at a given pH
In the redox titratations with Ech nearly all curves fitted
best to n¼ 2 Nernst lines As all titrations were
performed with H2/He mixtures, H2 is directly involved
in all reduction and oxidation reactions; hence n¼ 2 lines
are expected There is a notable difference in the results of
the redox titrations performed at pH 8: in the presence of
dyes the curve fitted best to a n¼ 1 Nernst line; when the
dyes were omitted the reaction was found to be n¼ 2
According to previous studies this could be due to the
artefacts caused by the redox dyes The titrations at
different pH values using two different methods show that
there is definite pH dependence of the midpoint potentials
of the Fe–S clusters responsible for the g¼ 1.92 and the
g¼ 1.89 signals (Fig 11) This effect was best observed in
the titrations in the absence of redox dyes at pH 8 and
pH 7 For the g¼ 1.92 signal the E0¢ value decreased by
53 mV per pH unit; this value was 62 mV per pH unit for
the g¼ 1.89 signal For the titrations in the presence of
redox dyes these values were 20 mV and 45 mV per pH
unit for the g¼ 1.92 and g ¼ 1.89 signals, respectively
This pH dependence for the proximal cluster (g¼ 1.89
signal) is in agreement with the pH dependence of the E0¢ value of the proximal cluster in standard [NiFe]-hydro-genases [42]
The values obtained for both signals were reasonably close to the theoretical value of)59 mV per pH unit for a redox reaction involving a stoichiometric amount of elec-trons and protons E0¢ values with such a large pH dependency are rare for [4Fe)4S] clusters with a classical Cys coordination [43–45] No firm conclusion is possible for the cluster causing the g¼ 1.96 signal The data indicate that the redox potential of this cluster is considerably lower than those of the other two clusters The existence of two [4Fe)4S] clusters with different midpoint potentials in one polypeptide is not unprecedented It was found in the ferredoxin of A vinosum [46]
The g-values (gz¼ 2.05 and gxy¼ 1.92) and pH dependence of )53 mV per pH unit of the g ¼ 1.92 signal, ascribed to one of the [4Fe)4S] clusters in the EchF subunit, is reminiscent of the g-values (gz¼ 2.054 and gxy¼ 1.922) and the pH dependence of)60 mV per
pH unit of the signal ascribed the cluster(s) N-2 of bovine complex I [22] There is a debate in the literature as to the precise location of this cluster N-2 [21,23,47–50] Ech contains only three [4Fe)4S] clusters and one of them (causing the g¼ 1.89 signal) is close to the Ni–Fe site and thus located in the EchC subunit Hence, in Ech the other two [4Fe)4S] clusters are in the EchF subunit which shows a very high amino acid sequence similarity to the TYKY subunit of the bovine complex I [23] This strengthens our earlier suggestion [23] that the Fe–S clusters in these subunits might be involved in an electron-transfer driven proton-pumping unit Further studies are required to verify this The data presented are a good starting point towards an understanding of the behaviour
of Fe–S clusters in proton-pumping [NiFe]-hydrogenases Point mutations of amino acid residues close to the several Fe–S clusters can give more insight into the mechanism of action At the same time the results obtained with Ech can
be helpful to a better understanding of similar studies in the field of complex I
A C K N O W L E D G E M E N T S
S P J Albracht is indebted to the Netherlands Organization for Scientific Research (NWO) for funding provided via the Section for Chemical Sciences R Hedderich acknowledges the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support.
Fig 10 Redox titrations of Ech in the presence of dyes and at different pH values The amplitudes of the g ¼ 1.92 (left panel) and g ¼ 1.89 signals (right panel) were plotted against the redox potential Solid curves represent Nernst curves with n ¼ 2 fitting to the data points at pH 6, 6.5 and
pH 7 At pH 8 the best fit was obtained with an n ¼ 1 curve The E 0 ¢ values are listed in Table 1.
Fig 11 Plots of the midpoint potentials (E 0 ¢) for both signals (g = 1.92
and g = 1.89) against the pH from the titration in the absence of dyes
(left panel) and from the titration in the presence of dyes (right panel).
The values used are those listed in Table 1.
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