Originally published In Press as doi:10.1074/jbc.X200002200 on August 6, 2002
J. Biol. Chem., Vol. 277, Issue 41, 37967-37972, October 11, 2002
REFLECTIONS
Bioinorganic Chemistry: A New Field or Discipline? Words,
Meanings, and Reality
Helmut
Beinert
From the Institute for Enzyme Research and Department of
Biochemistry, College of Agricultural and Life Sciences, University of
Wisconsin, Madison, Wisconsin 53726-4087
 |
INTRODUCTION |
To the uninitiated it may seem that bioinorganic chemistry is
a new field although, on the other hand, reports on metals bound to
proteins or enzymes date way back into the 19th century and may
probably be found in earlier centuries if we replace the terms "proteins" and "enzymes" with "animal or plant tissues."
Potassium ferricyanide was prepared from blood, McMunn described what
he called histohematins (now cytochromes) in tissues, Hoppe-Seyler made
spectroscopic investigations on hemoglobin, G. Bertrand worked on what
he called an oxidase from plant tissues, which he named laccase; he
recognized that it was a metal protein and even proposed that the metal
was a "coenzyme," with which he may have been the first to propose
the idea of a catalytic metal protein. Spitzer drew attention to the
involvement of iron bound to protein or nucleic acid nitrogen in tissue
respiration, Warburg and Keilin and their collaborators described
polyphenol oxidase, a copper protein, and ferritin was described as an
iron storage protein. In the 1930s Keilin and Hartree found copper in
cytochrome c oxidase. By the middle of that century, zinc
and molybdenum were discovered in enzymes, and non-heme iron was
recognized as a necessary component in mitochondrial preparations that
are active in substrate oxidation. Thus there is a long trail of
discovery of metals in proteins or in other components of living
creatures, metals that were required for their structure or function,
although details of what the functions implied were initially missing
and hard to come by.
At this point it seems worthwhile to define what is actually meant by
the terms "inorganic" and "organic" in connection with our
present theme or in the context of chemistry as a scientific discipline. Although the term "organic" will for many evoke the connotation that it has to do with life (the counterpart
"inorganic" then referring to lifeless matter) in chemistry
"organic" has come to mean merely pertaining to the chemistry of
carbon compounds. Inorganic, on the other hand, is generally perceived
as referring to the chemistry of metal compounds, whereas other
non-carbon non-metal elements are not specifically excluded. Because
metals of many kinds are found in living matter, e.g.
sodium, potassium, and calcium in considerable quantities, and because
all metals are subjects of inorganic chemistry, there must then, by
definition, always have been an inorganic component of biochemistry.
Thus, according to this reasoning, "inorganic biochemistry" and
"bioinorganic chemistry" certainly are no new subjects; rather they
may only be new words.
| |
The Hassle About an Acceptable Name for the Field of Our
Endeavors |
Nevertheless, the terms obviously had to be justified. The
editor of the first volume of Inorganic
Biochemistry, Gunter L. Eichhorn, says in 1974 in the
preface to the book: "Until recently, the title Inorganic
Biochemistry would have appeared paradoxical to most, and it
may even now appear so to many, because biochemistry sounds organic";
and this was about 30 years ago! As I have witnessed this period and
also some decades before that, I will briefly describe my experiences.
When I was a student in the 1930s, what Eichhorn calls paradoxical
would doubtless have been considered outright ridiculous, certainly in
Germany. When I studied chemistry there it was the great period of the
development of organic chemistry, synthetic and analytical, of natural
products chemistry, the time of the discovery of "factors" (some of
them later called vitamins), and I heard it said often in chemical
circles that biochemistry was in essence nothing but organic chemistry.
Actually even the word biochemistry was frowned upon, as it had
acquired some taste of quackery through the use of the word by some
eager promoters of what may today be called "alternative medicine."
The term "biochemistry," as far as I know, was in some sense
generally established when Sir Frederic Gowland Hopkins named his
department in Cambridge by that title in 1914, although the actual
building that had this name was only occupied in 1924. In Germany this
subject, "biochemistry," was not taught within a faculty of the
Naturwissenschaften but had the designation "physiological
chemistry" and was only accessible to medical students, and not to
chemistry students, as I was. When I inquired whether I might get an
exceptional permission, I was advised by friends in the faculty that
physiological chemistry was actually largely a "Gemurkse," which
means a messy business, going under the slogan, "Tierchemie ist
Schmierchemie," and I would do better to concentrate on chemistry. It
was only Felix Hoppe-Seyler who was influential enough to be allowed to
establish an Institute of Applied Chemistry within the Faculty of
Medicine at Tübingen, which then was soon renamed physiological
chemistry and was assigned to a separate Faculty of Science. As such it has survived Hoppe-Seyler for many years as well as his journal, which
now goes under the title "Biological Chemistry." Feodor Lynen was
the first to have a Max-Planck Institute for Biochemistry in Munich in
1954, after which biochemistry departments then started sprouting up
elsewhere. However, even in the United States the departments of
physiological chemistry only slowly disappeared.
| |
Development of the Field |
It is true that the major polymers in living matter are carbon
compounds, whereas the transition metals are only present in traces
(except e.g. for iron in the globins or in ferritin);
however, there is no life without transition metals, which are required as catalysts. From what was said above, there was a gap of about 30 years between the 1940s and the 1970s when there must have been a great
step forward in appreciating the significance of transition metals in
biology. I experienced the transition during that period most vividly
in two typical examples. I had the fortune to meet Edward Hartree
during a visit to Oxford and I asked him, of course, about his work
with Keilin in the 1930s on copper in cytochrome c oxidase,
which had also been the object of my studies (1). He said that they
were absolutely sure copper was there and was bound tightly to the
protein; they determined how much was there in comparison to heme, but
after that what else could they do? As spectroscopy was not applicable
to copper,1 they had no
method to tell them about its function. Thus Keilin decided not to
pursue this aspect further. I may call this the "Keilin-Hartree
dilemma"; almost all of metal biochemistry suffered from this
shortcoming. The second example came with my good fortune to be invited
to all seven sessions in the series on copper proteins, usually
referred to as the "Manziana Conferences," initiated and perpetuated by Bruno Mondovì of Rome, Jack Peisach of New York, and Bo Malmström of Göteborg and their colleagues
until 1995 (3). The 1972 to 1976 meetings still were under the spell of the Keilin-Hartree dilemma. The aspect of function largely eluded us.
In the copper field the advent of EPR clearly broke the ice. With this
technique (and thereafter with other spectroscopies) much more sense
could be made of the metal-to-protein stoichiometry and the electronic
absorption spectra that had been available so far. Now, all of a
sudden, those designations, such as CuA and CuB in cytochrome oxidase
or Type I, II, or III copper in ceruloplasmin assumed distinct
character. Things were not as simple with iron, because there a new
type of iron protein had to be discovered, the iron-sulfur (Fe-S)
proteins. The discovery predates somewhat the developments just
presented, but the more detailed exploration of this new field
approximately also falls into the same period. The coordination
chemistry of copper is relatively simple as compared with that of iron,
and it eventually took several different types of spectroscopies such
as Mössbauer (MB), electron nuclear double resonance (ENDOR),
x-ray absorption spectroscopy (XAS), extended x-ray absorption
fine structure (EXAFS), magnetic circular dichroism (MCD), and NMR,
which were just developed in those years to a state such that it became
feasible to apply them to biological material with relatively low
concentrations of the target structures and with limited stability.
| |
Approach between Disciplines |
In those copper protein meetings it was a most stimulating get
together of groups that had barely talked to each other before: the
chemical physicists that were trained in ligand field theory and were
on speaking terms with pioneers such as Carl Ballhaüsen (4) and
on the other side us, the enzyme chemists, used to getting our hands
dirty with awkward messes of animal, plant, or bacterial origin, from
which our objects had to be purified. We could not understand our data
without the wisdom of the spectroscopists, and they were anxious to get
their minds on the challenging and fascinating problems in metal
coordination that nature had to offer. This fortunate mutual approach
between these disciplines (the coordination chemists, the
spectroscopists or chemical physicists, and the biochemists) took place
in that 10-20-year span starting around the 1970s. There followed a
period of intensive research on the functions of these proteins, and,
no wonder, enzymes played a dominant role in this as compared with
other metal proteins that serve other roles with the result that
biological inorganic chemistry was often identified with the
biochemistry of metal enzymes. This, of course, led to some resentment
(5) in the ranks of those interested in metal proteins with other
functions such as transport, as e.g. hemocyanins, or metals
involved in geological processes or stabilization of biological
structure (6). In this period as more structural and spectroscopic data became known, chemists started taking considerable interest in complex
biological structures such as hemocyanin for instance or polyphenol
oxidases, exploring their copper-oxygen chemistry. This then led to
similar investigations on iron-oxo-systems, which eventually opened up
the whole new field of Fe-oxo-proteins (7). A strong relationship of
the chemistry of the metal-oxygen reactions to those involving free
radical mechanisms soon became apparent (8). A similar relationship was
found in the Fe-S protein field (9).
During that period, starting with the 1970s, the flood of volumes on
inorganic biochemistry, bioinorganic chemistry, metals in biological
systems or whatever they were called started pouring out and there soon
was also a Journal of Inorganic
Biochemistry, starting in 1972, joined more recently, in
1996, by the Journal of Biological
Inorganic Chemistry.
A particularly good example of the rapid progress in that period is the
role that x-ray crystallography played in the meeting series on copper
proteins that we have followed above. At the 1979 meeting we all
listened with awe to the only crystallographer present, Hans
Freeman of Sydney, who showed the structure of poplar plastocyanin,
one of the smallest blue copper proteins containing a single copper
ion. The data on the immediate environment of the copper confirmed the
conclusions drawn by the spectroscopists. At the 1985 meeting, Freeman
presented further detail such as structures of reduced plastocyanin and
of plastocyanins from different plant sources. Then, at the 1990 meeting, there were five crystallographers in the audience who
presented three new structures, namely those of the considerably more
complicated multicopper enzyme ascorbate oxidase, bacterial nitrite
reductase, and galactose oxidase. Finally, at the 1995 meeting there
were presented such impressive accomplishments as the structure of beef
heart cytochrome c oxidase, of human ceruloplasmin, and
amine oxidase of Escherichia coli. Of course, it must also
be mentioned that it was not only the development of crystallographic
methods and of more efficient light sources, it was also the progress
in the preparation of proteins in high purity and quantity and the
possibility of introducing new groups or exchanging amino acids (all
this via molecular genetic procedures) that contributed to the
remarkable advances. An example here is the preparation and structure
determination of the CuA module of cytochrome c oxidase
through such approaches (10).
| |
Development and Applications of Spectroscopies |
Similar advances were made in other areas using spectroscopy
with radiation all over the range of energies from 
rays to radio
frequencies. As my personal experience was mainly with spectroscopy, I
may be forgiven if, among examples, I will mainly draw on those that I
was directly involved in or that are in my field of interest.
Before we leave the realm of high energy radiation, we must mention the
relatively recent development of x-ray absorption spectroscopy, which
became feasible when sufficiently powerful beam lines became more
generally available at the reactor sites. Various features of these
spectra have attracted attention: XANES, x-ray absorption near edge
structure and more recently also pre-edge structure, and EXAFS, from
which distances to neighboring nuclei can be determined or estimated,
or certain types of nuclei can be excluded, depending on the quality of
the spectra. EXAFS, for instance, has played a decisive role in the
discovery and structure determination of the 3Fe cluster of Fe-S
proteins (11), and particular attention has been paid recently to the
pre-edge features in XANES (12), as it can furnish quantitative
information on the degree of covalency of metal-ligand bonds (13). This
has been successfully accomplished for Fe-S proteins and has given new
insights into the electronic structure of Fe-S proteins.
After this consideration of x-ray spectroscopy in connection with the
discussion of the development of x-ray diffraction and crystallography,
I will now briefly mention examples of the successful use of
spectroscopy at other frequencies, starting from the low energy end.
After a slow start, as far as application to proteins goes, NMR has
undergone a very impressive development after the introduction of two-
and higher-dimensional techniques, in combination with elaborate
pulsing techniques and Fourier transform analysis, particularly also by
the use of isotopes of a nuclear spin different from that of the
naturally occurring atoms, such as 2H, 15N,
13C, 17O, and 57Fe. For use with
metal proteins the exploration of "paramagnetic NMR," that is NMR
on paramagnetic substances, has had great success (14) and has now
become a routine procedure and the method of choice for answering
specific questions. When the sequence of a protein is known and
preferably also the three-dimensional structure around the metal site,
the unpaired spin density on specific atoms can be determined. It has
even been possible to observe the migration of spin density between
sites (15). Moving on to the millimeter and centimeter range, EPR has
been mentioned above as one of the first decisive tools in approaching
the aspects of the function of copper proteins. It has played the
decisive role in the discovery of Fe-S proteins and the exploration of
the electronic structure and other properties of these proteins (16).
The hybrid methods such as ENDOR and ESEEM or optical detection
of magnetic resonance are making use of the relatively slow relaxation
of electron spins, so that saturation with incident radiation
(microwaves in EPR) may occur, which can then be relieved through
energy transfer by exciting neighboring atoms with other frequencies
(radiowaves in ENDOR or ESEEM). Again, by the use of isotopes of
different nuclear spin, very specific information can be obtained on
the kind of neighboring atoms, on interatomic distances, and even on
the mutual orientation of interacting species. Thus, for instance, we
have been able to determine that in the 4Fe cluster of aconitase the
specific iron atom (Fea) that has no cysteine ligand has a hydroxyl bound in the absence of substrate, which becomes protonated on
addition of substrate to the enzyme (17). We could also show with this
enzyme and with substrate labeled with 17O or
13C in different positions that the 
-hydroxyl of
isocitrate and the 
-carboxyl of citrate bind to Fea
(18).
The ESEEM method is applicable for the detection of more distant
neighboring atoms. Again pulse and Fourier transform techniques are
required in this instance. An example of the combined use of both ENDOR
and electron spin echo envelope modulation (ESEEM) is the
identification of the sequence of radicals formed in the conversion of
-lysine to -lysine by 2,3-lysine aminomutase (19). A condition
necessary for all the work mentioned here is that a measurable EPR
signal can be observed. In the cases cited it was the EPR signal of a
reduced [4Fe-4S] cluster or of a free radical.
Resonance Raman (RR) spectroscopy is based on the enhancement of
ordinarily observed Raman lines by a transition metal ion present in a
molecule. This technique is therefore able to provide specific
information concerning the ligands of the metal center and their
position with respect to the metal. Again the use of specific isotopes
of different molecular mass, such as 2H, 13C,
15N, 18O, 34S, and 54Fe
is very helpful and decisive. For instance it has been argued early on
the basis of RR (20) that the newly discovered 3Fe cluster could not
have the [3Fe-3S] benzene-like ring structure first assumed but must
have a structure closely related to the 4Fe cluster, as was shown by
EXAFS and chemical analysis (11).
An impressive example of the analytical power of infrared spectroscopy
when applied to proteins was furnished when it was discovered that
hydrogenases have CO and CN ligands bound to their 2Fe cluster (21). It
has also been possible to detect subtle changes in substrates by
infrared (22).
Among the methods relying on magnetism the simplest may seem to be
direct measurement of magnetic susceptibility. However, it is
technically quite difficult to achieve the desired sensitivity. MCD and
more so yet VTMCD, namely variable temperature MCD, has taken a
prominent position for discriminating different components in
e.g. a protein containing a number of different heme groups. This technique furnished the clue in the determination of the different
Fe-S clusters in succinate dehydrogenase (23). It has also been useful
in the determination of the spin state of a substance when EPR is
ambiguous. An example here is the determination of the spin state of
the reduced [3Fe-4S]0 cluster (24).
At about the middle of the 1960s I was invited to give a lecture at the
Max-Planck Institute for Medical Research in Heidelberg. It was
there in the Physics Division where Rudolf Mössbauer as a graduate student had discovered the effect now bearing his name. Richard Kuhn, the director of the Institute, introduced me in his
unmistakable Viennese accent and his at such occasions usually somewhat
pompous way: "Warburg has given us, who are interested in biological
oxidations, the heme iron; you have now given us the non-heme iron"
(referring to my work on Fe-S proteins). There happened to be in the
audience Ekkehard Fluck, a young "Dozent" at the university, who
was one of the early explorers of the MB effect in chemistry; he jumped
up and commented that I should use MB to find out what these non-heme
iron compounds were. When I asked how much iron (and that was to be
57Fe) and in what volume was needed for this, his answer
made it clear that this was definitely not the method to use.
Nevertheless, it was not even 10 years before my friend Richard H. Sands and others (25) indeed successfully used MB on purified
ferredoxins of relatively low molecular weight (though not on
mitochondria yet), but it was then 20-30 years later that MB could be
applied with success to identify iron in whole bacterial cells (26) and
that specific compounds could be recognized and quantitatively determined (27). There was an effect of mutual stimulation, similar to
what I described above, when the first EPR data appeared but, of
course, at a much more advanced level. Although we biochemists learned
something about our proteins and enzymes, so did our challenge stimulate the spectroscopists to optimize the conditions and
particularly also to dig out from the literature applicable theoretical
concepts that could explain the observations, extend them, or even
develop new ones of their own. This led to a closer approach between
theoreticians and us at the bench, which turned out to be very
productive. Thus, for instance, the concept of
"spin-dependent delocalization (SDD)" (also called
"double exchange" or "resonance") was so clearly demonstrated,
first by the reduced 3Fe cluster [3Fe-4S]0 (28) and then
also for the 4Fe cluster that it is now one of the main features
considered important for understanding the electronic structure and
reactivity of these clusters. The concept was, of course, not new but
was hidden in mathematical equations and under designations that are
not easily understood. It is the merit of the colleagues whose names
appear in the given references that has brought these concepts to our
attention in terms understandable to us (29). The electronic structure
of the clusters with more than two iron atoms can now be understood in
terms of these interacting forces: 1) SDD, which favors a parallel spin
orientation and thus formation of mixed valence (MV) pairs with one
shared electron; 2) J, which favors antiparallel coupling as in the 2Fe
cluster; and 3) what is called vibronic coupling, which has to do with the symmetry of the surrounding protein environment (e.g. a
non-Cys ligand) and may favor one or the other spin arrangement. Cases for this are documented, with one of the simplest and most impressive examples being the 2Fe ferredoxin from a mutant of Clostridium pasteurianum in which one serine replaces one of the cysteine ligands of the native structure (30).
All the in some way related areas of endeavor mentioned above have, in
their own way, become specialty fields, encompassing already a
voluminous literature. Thus, definitions of fields are becoming
blurred, and we must recognize the fallacy of trying to categorize with
any rigidity while still preserving real meaning. Fig.
1 may represent a possible way to depict
the situation, as seen from the horizon of a biochemist. The fact that
biochemistry occupies a space larger than the others does not mean that
it is more important, but it was necessary for practical reasons, namely to provide enough space for demonstrating the overlap.
| |
FOOTNOTES |
Published, JBC Papers in Press, August 6, 2002, DOI 10.1074/jbc.X200002200
Address correspondence to: hbeinert@facstaff.wisc.edu.
1
The broad 830 nm absorption of cytochrome
c oxidase was only discovered in 1961 (2) and was probably
not observable with Keilin's microspectroscope.
| |
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INTRODUCTION
The Hassle About an...
