Metallobiochemistry [Part D] [No cover or index]

665 Pages·1993·33.82 MB·English

by  J. Riordan, B. Vallee

Preface
The scope of metallobiochemistry has greatly expanded in recent
years as ever more powerful techniques have been brought to bear on the
constituent elements that define and characterize the subject. Metallobio-
chemistry, Part A, Volume 851 of Methods ni Enzymology, focused on
progress in those areas in which early on there were major impediments to
growth and development. Without the tools to measure metals with suffi-
cient accuracy, precision, and sensitivity this scientific discipline could
not have existed. In addition, unless it were possible to distinguish be-
tween the metals present in a biological sample that belonged there (be-
cause nature intended them to be) from those that merely appeared by
accident (adventitious contamination), such metal analyses would have
been meaningless. Technology overcame these hurdles, and Part A de-
tailed the state of trace element analysis and the various approaches
employed by the metallobiochemist to avoid artifacts and achieve the
inorganic equivalent of microbiological sterility. It is a summary of the
critical methods that have helped place the subject on a solid foundation.
Metallobiochemistry, Part B, Volume 502 of this series, is devoted to
metallothionein and related molecules. It is unusual for a Methods ni
Enzymology volume to feature a single molecule, but the surge of interest
in metallothionein and its structural and possibly functional relationship
to DNA-binding proteins suggested that such a volume would be timely
and useful. It also seemed appropriate to stress that not all metal-contain-
ing biological molecules are metalloenzymes or electron-transport pro-
teins.
Parts C and D, Volumes 622 and 227, respectively, return to the theme
of methods that have contributed to and are emerging as important factors
in the advancement of the field. These methods embody concepts that had
their origin about the time of the almost forgotten Sumner-Willst~itter
controversy of the 1920s. Proteins, it was claimed, could hardly serve as
specific biological catalysts if they were little more than nondescript col-
loids. Metal ions would prove to be the real actors on the enzymatic stage.
Authority prevailed until the crystallization of urease seemingly dis-
patched the metal dogma to oblivion. Despite the extended protests of
interest-vested diehards, protein chemistry became inextricably associ-
ated with enzymology and metals fell out of fashion. (Ironically, urease
turned out to be a nickel enzyme.)
Biochemists who had witnessed this metal-induced brouhaha were
understandably reluctant to resurrect the idea that metals might have
something to do with biological catalysis. Anyone wishing to make the
case would have to have persistence along with persuasive and unassail-
ix
X ECAFERP
able analytical data. Only through scrupulous attention to detail was it
possible in those neonatal days of metallobiochemistry to gain the accep-
tance that allowed the field to grow and flourish.
Despite its shortcomings the metal-cum-colloid view of catalysis did
have one rather appealing feature: the metal would have unique proper-
ties among all the atoms of the protein and perhaps these could be ex-
ploited to gain important information. The metal could serve as a beacon
to guide the investigator searching for an active site. It could also be a
signal, either of the detailed steps of catalysis or of any other biological
function with which the metal might be associated. Emission spectros-
copy proved the significance of a metal-derived signal in principle, but
was rather inconsiderate of the protein. Hence, attention shifted along
with wavelength to absorption spectroscopy whereby it became possible
to view the functional heart of a metalloenzyme directly. This window on
the world of metallobiochemistry revealed unprecedented spectral fea-
tures clearly indicative of an unusual coordination environment and likely
characteristic of a catalytic site.
Not all metals lend themselves to absorption spectroscopic investiga-
tion. Zinc, one of nature's most recurrent participants, is notoriously shy
in this regard. Other metals are more expressive and revealing when
viewed by alternative techniques. In these two volumes (226 and 227) we
have assembled a broad representation of the physical and spectroscopic
methods now available that can be useful for examining metals in biologi-
cal systems and for probing their environments in metalloproteins and
metalloenzymes. These approaches, while by no means all-inclusive, ex-
emplify the wide variety of tools and the level of sophistication currently
being applied to extract both the nuances and the general principles of
metallobiochemistry pertinent to these systems.
We are extremely grateful to our contributors for their willingness to
participate in this endeavor. They have made a concerted effort to de-
scribe techniques in ways that would be most beneficial to the reader. The
chapters differ from the more typical ones in this series in that they
identify principles underlying a particular method, the kinds of questions
that can be addressed, and the ways to interpret results. Step-by-step
instructions were not practical in most cases, and generally the objective
has been to provide a sense of what can be accomplished. It required
more description than anticipated for most of the topics, and this necessi-
tated two volumes instead of one. We appreciate the understanding of our
colleagues at Academic Press and we thank them again as well as all the
contributors for making this such a pleasant experience.
JAMES F. RIORDAN
BERT L. VALLEE
1 2D NMR FO CITENGAMARAP SNIETORPOLLATEM 1
1 Two-Dimensional Nuclear Magnetic Resonance
of Paramagnetic Metalloproteins
By ANT6NIO V. XAVIER, DAVID L. TURNER, and HELENA SOTNAS
Introduction
Depending on the interactions between nuclear spins and unpaired
electrons, the parameters of the nuclear magnetic resonance (NMR) spec-
tra of paramagnetic molecules may be drastically different from those of
diamagnetic onesl-5: the relaxation times, ~T and T 2, may be shortened,
and the chemical shifts, 8, can be changed. For nuclei that are more than
a few bonds away from the paramagnetic center, their interaction with the
unpaired electron is purely dipolar and depends on geometrical functions
(including Curie relaxationS-7); thus, they provide important structural in-
formation.
Although the relaxation induced by the unpaired electron can be such
that the signals from resonating nuclei are so broad as to be undetectable by
NMR, the paramagnetically induced shifts generally result in an increase in
spectral resolution. Because the correlation time for the unpaired electron
relaxation, zs, is the dominant parameter contributing to the relaxation of
magnetic nuclei in a paramagnetic macromolecule, the application of NMR
in the study of these molecules is limited to those cases in which zs is not
too long. Several examples of the successful use of one-dimensional (ID)
NMR in the study of paramagnetic molecules are available, 4 as well as ex-
amples of structural studies in which paramagnetic centers naturally pres-
ent, extrinsically added, or introduced by isomorphous replacement of spec-
troscopically unsuitable ones are used to obtain geometrical informa-
tion °I-8 or just to improve resolution and to assist in spectral assignments.
i R. A. Dwek, R. J. P. Williams, and A. V. Xavier, in "Metal Ions in Biological Systems"
(H. Sigel, ed.), p. 61. Dekker, New York, 1974.
2 G. N. La Mar, in "Biological Applications of Magnetic Resonance" (R. G. Shulman,
ed.), p. 305. Academic Press, New York, 1979.
3 j. D. Satterlee, Annu. Rep. NMR Spectrosc. 17, 79 (1986).
4 I. Bertini and C. Luchinat, "NMR of Paramagnetic Molecules in Biological Systems."
Benjamin/Cummings, New York, 1986.
5 I. Bertini, L. Banci, and C. Luchinat, this series, Vol. 177, p. 246.
6 M. Gueron, J. Magn. Reson. 19, 58 (1975).
7 A. J. Vega and D. Fiat, Mol. Phys. 31, 347 (1976).
8 C. D. Barry, A. C. T. North, J. A. Glasel, R. J. P. Williams, and A. V. Xavier, Nature
(London) 232, 236 (1971).
thgirypoC © 3991 by Academic Press, Inc.
SDOHTEM NI ,YGOLOMYZNE VOL. 722 llA rights of noitcudorper ni any form reserved.
2 PROBES OF METAL ION ENVIRONMENTS 1
The advent of two-dimensional (2D) NMR techniques, which has
greatly enhanced the range of potential applications of NMR spectroscopy
to the study of macromolecules, 11 posed a further constraint in possible
applications to the study of paramagnetic molecules. In fact, the 2D NMR
technique can reveal scalar and dipolar (or chemical exchange) interac-
tions, through the observation of connectivity signals (cross-peaks) be-
tween different nuclei (or the same nuclei but in different chemical environ-
ments) that develop during specially designed mixing and evolution
periods. Because the frequency of labeling decays with the relaxation rate
of the interacting nuclei, there is a stringent time scale for the useful
application of 2D NMR to the study of paramagnetic molecules. Thus,
specific conditions must be used in order to optimize the acquisition of
the information required.
The application of 2D NMR to paramagnetic proteins has grown
exponentially, lagging just a few years behind the growth of applications
to diamagnetic systems. For example, in 1986 Wuthrich 11 provided the
first comprehensive account of 2D methods in protein NMR, Satterlee 3
mentioned that this technique should have application for paramagnetic
systems in the future, and Bertini and LuchinaP mentioned just one
example of a 2D NMR study of paramagnetic proteins. 21 The nuclear
Overhauser effect spectroscopy (NOESY) 31 sequence was first applied
to paramagnetic proteins in order to detect chemical exchange be-
tween two paramagnetic forms 21 (see Fig. )1 and between a diamag-
netic and paramagnetic form called exchange correlated spectroscopy
(EXCTSY)14, making it possible to assign resonances in the paramag-
netic (oxidized) spectrum from independently assigned resonances in
the diamagnetic (reduced) one. Of course, those experiments also
provided information about nuclear Overhauser effects (NOEs), but the
technique was not widely used for paramagnetic proteins until 51.8891
Similarly, correlation spectroscopy (COSY) 61 was used to detect scalar
couplings between aromatic protons 71 and between a-CH and NH
9 I. D. Campbell, C. M. Dobson, R. J. Williams, and A. V. Xavier, Annu. N. Y. Acad. Sci.
222, 361 (1973).
0~ R. A. Dwek, "NMR in Biochemistry." Oxford Univ. Press (Clarendon), Oxford, 1973.
H K. Wuthrich, "NMR of Proteins and Nucleic Acids." Wiley, New York, 1986.
21 H. Santos, D. L. Turner, A. V. Xavier, and J. LeGall, J. Magn. Reson. 59, 771 (1984).
31 j. Jeener, B. H. Meier, P. Bachmann, and R. R. Ernst, J. Chem. Phys. 71, 4546 (1979).
41 j. Boyd, G. R. Moore, and G. Williams, J. Magn. Reson. 58, 115 (1984).
51 S. J. McLachlan, G. N. La Mar, and K.-B. Lee, Biochim. Biophys. Acta 957, 430 (1988).
61 W. P. Aue, E. Bartholdi, and R. R. Ernst, J. Chem. Phys. 64, 2229 (1976).
7~ G. Williams, G. R. Moore, R. Porteous, M. N. Robinson, N. Soffe, and R. J. P. Williams,
J. Mol. Biol. 183, 409 (1985).
81 A. J. Wand, H. Roder, and S. W. Englander, Biochemistry 25, 1107 (1986),
1 2D NMR oF CITENGAMARAP SN1ETORPOLLATEM 3
-15
~( ~M s
-20
0 o
-25
nr0!D
nrpp . . . . 5'2 . . . . 20 ' . . . . 51'
FIG. .1 NOESY spectra of partially oxidized Desulfooibrio oulgaris cytochrome c 3 show-
ing cross-peaks resulting from intermolecular electron transfer. The sample was a 2 mM
solution in 2H20 at 892 K. The spectrum was recorded on a Bruker (Karlsruhe, Germany)
AMX500 (500 MHz) spectrometer using a mixing time of 52 msec with the transmitter
frequency set at the position of the residual water line, which was suppressed by presaturation
for 2 sec. Pure absorption peaks were obtained using time proportional phase incrementation
(TPPI) with 2048 points in 2t and 215 increments in tl. The data were zero-filled to 2048 ×
4201 points, and a window function for Lorentzian to Gaussian line shape transformation
was applied in 2t and a cosine-square function in ~t prior to the 2D Fourier Transform (FT).
A peak connecting resonances from molecules in which two and three hemes are oxidized
is labeled with its specific assignment (c.f. Ref. 72).
protons 8~ far from the paramagnetic centers and later to assign protons
close to an oxidized heme. 91 A variety of related methods including
double quantum filter (DQF)-COSY 12,°2 and 2D total correlation spec-
troscopy (TOCSY) 22 (see Fig. 2) were in use by 1990. 72-32
91 H. Santos and D. L. Turner, FEBS Lett. 226, 971 (1987).
o2 M. W. Edwards and A. Bax, J. Am. Chem. Soc. 108, 819 (1986).
12 N. Muller, R. R. Ernst, and K. Wuthrich, J. Am. Chem. Soc. 108, 6487 0986).
22 L. Braunschweiler and R. R. Ernst, J. Magn. Reson. 53, 125 (1983).
32 y. Yamamoto, A. Osawa, Y. Inoue, R. Chuj6, and T. Suzuki, FEBS Lett. 247, 362 (1989).
42 y. Feng, H. Roder, and S. W. Englander, Biophys. J. 57, 51 (1990).
52 S. D. Emerson and G. N. La Mar, Biochemistry 29, 1545 (1990).
4 PROBES OF METAL ION ENVIRONMENTS 1
11
10
I !
"I
.I
41
15
mpp
0'I 5 ~(
ppm 5'1
FIG. 2. TOCSY spectrum of Methylophilus methylotrophus ferricytochrome c", 2 mM
in 2H20 at 300 K. A 50 msec mixing time was used with a power level of 01 kHz and WALTZ
modulation for spin locking. An exponential window function was used in t 2, and the
remaining conditions for data recording and processing were the same as those given in the
legend to Fig. .1 The complete spin systems of one of the axial histidine ligands (H) and
one of the heme propionates (P) are indicated by boxes (c.f. Ref. 73).
Heteronuclear COSY was applied to paramagnetic systems earlier
than homonuclear proton experiments. Correlation of C~1 and IH shifts
of groups far from the paramagnetic center was used with C31 enrichment
in 1983, 8z but correlations between H1 and C31 in natural abundance were
used to assign methyl groups of an oxidized heme as early as 1986 9z (Fig. 3).
Any technique that can be applied to a diamagnetic protein can (and
will) be used to study paramagnetic proteins. Indeed, large sections of
the polypeptide chain that are remote from the paramagnetic center are
62 S. C. Busse, S. J. Moench, and J. D. Satterlee, Biophys. J. 58, 45 (1990).
72 L. P. Yu, G. N. La Mar, and K. Rajarathnam, J. Am. Chem. Soc. 112, 9527 (1990).
82 T. M. Chan and J. Markley, Biochemistry 22, 5996 (1983).
92 H. Santos and D. L. Turner, FEBS Lett. 194~ 73 (1986).
1 2D NMR OF PARAMAGNETIC METALLOPROTEINS 5
t. ~
PROTON °'~
~-e
~o
I
~ E i
o
W E
o
I
0 £ o I
oi
C~
n"
W ~.e
_U
~z
.~'!~
°
i
~ ;i~ ~-
.~. ~'~
i
.9
6 PROBES OF METAL ION ENVIRONMENTS 1
essentially indistinguishable from their equivalents in diamagnetic sys-
tems. We therefore concentrate on nuclei with large dipolar or Fermi
contact shifts and/or relaxed by paramagnetic metal ions. For most pur-
poses the nuclei outside a sphere or approximately 0.75 nm from the
paramagnetic center can be considered as "diamagnetic."
Before considering the information that may be obtained from two-
(three- or four-) dimensional experiments, it should be stressed that the
same information can always be obtained from a series of one-dimensional
experiments. These may be selective analogs of the 2D experiments or,
in the case of the NOE, the development of a equilibrium with a resonance
which is selectively and continuously saturated. Of course, this one-dimen-
sional NOE experiment produces a larger effect than the dynamic process
of relaxation of the complete spin system which occurs in NOESY, which
is a dynamic NOE experiment.
The effective sensitivity (signal-to-noise ratio, S/N) per unit time of a
two-dimensional experiment may be expressed as °3
,2*T
which depends on the effective decay rate, and the length, ,2"7 of the
free induction decay sampled in each sequence as well as the decay rate,
,2#T
during the evolution period, which is incremented up to a maximum
length .1"7 One-dimensional experiments only depend on the first term in
the equation, as can be seen by letting 1"7 tend to zero, so they are less
affected by more rapid relaxation. However, each selective one-dimen-
sional experiment probes the interactions of a single nucleus, whereas
a two-dimensional experiment provides information about all possible
interactions simultaneously. It is therefore a balance between the second
term of the equation and the number of interactions to be observed that
governs the choice of approach.
These may not be the only considerations because it is often convenient
to have cross-peaks from known interactions present to provide a refer-
ence for the same sample and spectrometer conditions. It is clear, how-
ever, that if the interactions of a single resonance are sought, then a one-
dimensional experiment will be far more efficient. °3,62
Experimental Considerations
Having decided that the system under investigation is sufficiently com-
plex to warrant the use of 2D methods, we may now consider which
o3 D. L. Turner, J. Magn. Reson. 61, 28 (1985).
1 2D NMR FO CITENGAMARAP SNIETORPOLLATEM 7
methods are the most useful. The simplest experiment for detecting scalar
(J) couplings between nuclei is COSY, which may be applied to homonu-
clear or heteronuclear systems. The main advantage of COSY is that
the magnetization transfer is effected almost instantaneously by a radio
frequency pulse so that there is no loss of signal through relaxation. The
main disadvantage is that the cross-peaks comprise several components
of opposite phase which will cancel each other out if the intrinsic line
width is large with respect to the coupling constant.
A second complication, which is shared by most other 2D experiments,
is that the spectrum correlates the frequency (with respect to the transmitter
or reference frequency) of a resonance in F 2 with another in F~, together
with the negative of its ~F frequency. If the spectral width in the second
dimension (FI) is the same as that in F2, then the positive and negative
frequency patterns will overlap. This may be resolved by canceling one
set of signals by phase cycling (usually the positive Fl frequencies, called
N-type selection), which is undesirable because information is being elimi-
nated, or by separating the two patterns and then recombining them so
that they are superimposed. The latter approach will also generate "pure
phase" signals, that is, signals with simple absorption, or dispersion cross
sections in both dimensions. A number of methods can be used to achieve
this: States, ~3 TPPI, 23 and hypercomplex 33 procedures are most common.
They share the same principles but differ in the routing of data such that
various artifacts may appear in different places with respect to the true
COSY spectrum. Most groups use absolute value spectra because of the
cancellation of pure absorption cross-peaks that occurs when the lines
are broad. 63-43'72 This is unwise because all of the intensity at the center
of the cross-peak comes form the dispersion mode (the absorption mode
gives a zero signal on cross sections through the center, regardless of
linewidth), so that the absolute value spectrum will degrade the signal-
to-noise ratio first by mixing the independent noise traces from the ab-
sorption and dispersion components and, second, by rendering it purely
positive. The best procedure for broad lines is to use a matched filter,
sin(TrJt) exp(-t/Tz), and to phase the cross-peaks correctly to be purely
dispersive. This has the additional advantage of producing pure absorption
and therefore narrow peaks on the diagonal. If the spectrum is poorly
13 D. J. States, R. A. Haberkorn, and D. J. Ruben, J. Magn. Reson. 48, 286 (1982).
23 D. Marion and K. Wuthrich, Biochem. Biophys. Res. Commun. 113, 769 (1983).
33 L. Muller and R. R. Ernst, Mol. Phys. 38, 369 (1979).
43 I. Bertini, F. Copozzi, C. Luchinat, and P. Turano, J. Magn. Reson. 95, 442 (1991).
53 j. D. Satterlee, D. J. Russell, and J. E. Erman, Biochemistry 30, 9072 (1991).
63 K. A. Keating, J. S. de Ropp, G. N. La Mar, A. L. Balch, F.-Y. Shiau, and K. M. Smith,
lnorg. Chem. 30, 3258 (1991).
8 PROBES OF METAL ION ENVIRONMENTS 1
digitized, there may be some advantages in examining an absolute value
plot of the spectrum as well. However, many pulse sequences for "mag-
nitude" or "absolute value" COSY employ N- or P-type peak selection,
and these should never be used because they lead to loss of half of the
signal.
Narrow diagonal peaks allow cross-peaks to be observed between
resonances with small separations. This is the principal purpose of the
double quantum filter in DQF-COSY, together with the elimination of
singlets that may arise from the solvent or from impurities. The elimination
of singlets is not usually very important, but the narrow diagonal is
achieved at the cost of one-half of the cross-peak intensity. A pure dis-
persion COSY is therefore preferable. The same applies to the
"ISECR-COSY ''37 experiment, which incorporates refocusing delays of
the order of I/Z/for the purpose of bringing the components of the cross-
peaks into the same phase: the signal is reduced by a further approximately
50 msec of relaxation for very little gain.
Extended mixing periods can be useful, however, to find resonances
that belong to the same spin system (a set of coupled nuclei within a single
side-chain or prosthetic group) but do not couple directly with each other.
Experiments of this type include relayed COSY 83 and TOCSY. 93'22 The
first involves a number of COSY-type magnetization transfer steps with
refocusing delays that can be optimized for a particular series of coupling
constants. The TOCSY experiment uses a somewhat different principle
insofar as the radio frequency field is applied continuously during the
mixing period and creates a series of cross-peaks to other nuclei of the
same spin system that have all their components of the same phase. The
transfer of magnetization between nuclei requires times of the order of
1/2J and is an oscillatory process, so some cross-peaks of the spin system
may be weak for a single mixing time. In principle, a full set of cross-
peaks between, for example, an amide proton and the shifts of all the
protons in a particular residue may be observed, which may well allow a
primary assignment to be made directly. There is a potential disadvantage
in that correlations caused by cross-relaxation (NOEs) may then also
occur, and these are of opposite sign to the cross-peaks generated by J
coupling. Two protons may be coupled and also cross-relax, so that the
cross-peak between them may be partially canceled. When short mixing
times (<50 msec) are used to optimize an experiment by balancing the
buildup of the cross-peaks against the loss of signal through relaxation,
73 S. Talburi and N. A. Scherage, J. Magn. Reson. 86, 1 (1990).
83 G. Eich, G. Bodenhausen, and R. R. Ernst, J. Am. Chem. Soc. 104, 3731 (1982).
93 A. Bax and D. G. Davis, J. Magn. Reson. 65, 553 (1985).