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REVIEWS

TIBS 25 – MARCH 2000

Function of the
ubiquitin–proteasome pathway
in auxin response
William M. Gray and Mark Estelle
The plant hormone auxin regulates many aspects of growth and development. Despite the importance of this hormone, the molecular basis for
auxin action has remained elusive. Recent advances using molecular
genetics in Arabidopsis have begun to elucidate the mechanisms involved
in auxin signaling. These results suggest that protein degradation by the
ubiquitin pathway has a central role in auxin response.

related modifier; also known as PIC1/
Ubl1/Sentrin, and Smt3p in yeast) is
approximately 20% identical to ubiquitin
and is conjugated to several proteins, including RanGAP1, PML and IkB in mammals and the septin proteins of budding
yeast8–11. In several cases, modification
by SUMO-1 appears to regulate subcellular localization of the target protein. The
RUB1 (related to ubiquitin 1) family

(NEDD8 in mammals) of Ubls is 50–60%
identical to ubiquitin. Less is known
about the function of these proteins.
Genetic analysis of the yeast cell cycle
and the Arabidopsis auxin-response pathway suggests that RUB1/NEDD8 modification might regulate the activity of a
subset of E3 ubiquitin ligases (see
below).

Approaches to studying auxin action
SELECTIVE PROTEIN DEGRADATION
has emerged as a regulatory mechanism
for a wide variety of cellular processes.
In eukaryotic cells, the ubiquitin system
is a major pathway for regulated protein
degradation. Ubiquitin is a highly conserved 76-amino-acid protein that covalently modifies target proteins, marking
them for degradation by the 26S proteasome. This pathway regulates key biological processes such as cell division,
metabolism, immune response and
apoptosis1.
Recent studies with Arabidopsis
thaliana have revealed that the ubiquitin

proteolytic system plays a central role in
the auxin-response pathway. The plant
hormone indole-3-acetic acid (IAA or
auxin) controls many aspects of plant
growth and development. Some of the
best-characterized examples are tropic
growth responses, stem elongation, lateral branching of roots and shoots,
and vascular development2. These
processes are controlled by auxin-mediated
changes in cell division, cell expansion
and cell differentiation. Given the prominent role of auxin in these basic cellular
events, it is hardly surprising that plant
biologists have long been intrigued by
this hormone and have compiled an enormous amount of physiological data concerning the responses of plants to IAA.
Nonetheless, fundamental aspects of
auxin biology such as auxin biosynthesis,
perception and response are still poorly
W.M. Gray and M. Estelle are at the Institute
for Cellular and Molecular Biology, Section of
Molecular, Cellular, and Developmental

Biology, University of Texas at Austin, Austin,
TX 78712, USA.
Email: mestelle@mail.utexas.edu

understood. This review will focus on
recent advances that implicate the
ubiquitin pathway in the auxin response.

The ubiquitin-conjugation pathway
Ubiquitin conjugation involves an enzymatic cascade in which ubiquitin is
first activated by the formation of a thiolester bond between its C terminus and a
cysteine residue within the ubiquitinactivating enzyme (E1). Ubiquitin is then
transesterified to a member of a family
of ubiquitin-conjugating enzymes (E2).
Finally, with the assistance of a ubiquitin
ligase (E3), ubiquitin is covalently attached to an e-NH2 group of a lysine
residue within the substrate protein3.
Ubiquitin ligases have a crucial role in
determining substrate specificity. E3 enzymes are very diverse and the least
understood components of the ubiquitinconjugation pathway. In some cases, the

E3 forms a catalytic intermediate with
ubiquitin4, whereas in others, the major
function of the E3 might be to bring the
E2 and the substrate into close proximity5. Reiteration of these reactions using
a specific lysine residue within the conjugated ubiquitin results in the generation of a polyubiquitin chain. The 26S
proteasome recognizes this chain and
degrades the tagged protein, releasing
free ubiquitin in the process.
In recent years, a number of ubiquitinlike proteins (Ubls) have been identified.
These proteins are conjugated to a lysine
residue of target proteins by a mechanism very similar to ubiquitin conjugation6,7. However, in marked contrast to
ubiquitination, Ubl conjugation does not
generate a polyUbl chain and does not
appear to affect target protein stability.
The SUMO-1 protein (for small ubiquitin-

0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.

Two strategies have been employed
to identify genes involved in auxinmediated growth and development. The

first approach is the recovery of mutants that exhibit resistance or reduced
response to applied auxin. The second
strategy has been to identify genes that
are rapidly induced by auxin and then
link these to factors acting upstream in
the response pathway. Both of these approaches have been successful, and it
appears that the two might be about to
converge on the ubiquitin pathway.

Studies of auxin-response mutants
In Arabidopsis, several mutants have
been isolated that display diminished response to auxin12. Genetic analysis suggests that four of these genes (AXR1, TIR1,
AXR4 and SAR1) act in the same or overlapping pathways. Loss-of-function mutations in AXR1, AXR4 and TIR1 confer
diminished auxin response, and double
mutant combinations between these
genes display synergistic interactions13–15.
Phenotypic analyses indicate that
AXR1 (auxin resistant 1) plays a central
role in auxin signaling (Fig. 1a). axr1 mutants exhibit defects in essentially all
processes thought to be mediated by

auxin, including meristem function,
tropic growth responses and cell elongation. Additionally, axr1 plants exhibit
reduced expression of the Aux/IAA and
SAUR (for small auxin up RNA) auxininducible genes. The AXR1 protein is related to the N-terminal half of ubiquitinactivating enzyme16. AXR1 interacts
with the ECR1 (E1 C-terminus-related 1)
protein to form a bipartite enzyme that
activates the RUB Ubl proteins17.
Arabidopsis contains at least three members of the RUB family. RUB1 and RUB2

PII: S0968-0004(00)01544-9

133

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(a)

(b)

ATP

S

AMP + PPi

S

O C
H N

ECR1
AXR1

RCE1

AtCUL1
Ti BS

Figure 1

The AXR1 (for auxin resistant 1) auxin-response gene encodes a subunit of the RUB1 (for
related to ubiquitin 1)-activating enzyme. (a) 35-day-old wild-type (wt) and axr1-12 plants.
Bar, 5 cm. (b) The heterodimeric AXR1–ECR1 (for E1 C-terminus-related 1) enzyme
activates RUB1 (orange) for conjugation by forming a thiolester bond with RUB1. RUB1 is
subsequently transesterified to the RUB1-conjugating enzyme 1 (RCE1), which covalently
attaches RUB1 to the substrate AtCUL1, an Arabidopsis cullin.

E2

Sk

p1

Rbx1

Cul1

F

F

F

Ti BS

Figure 2
The SCF (for Skp1p, Cdc53p/cullin and Fbox protein) ubiquitin-ligase model.
Skp1, Cul1 and Rbx1 comprise the ‘core’
SCF complex (top), which interacts with
an E2 enzyme. F-box proteins (middle)
act as adaptor subunits that recruit
specific substrates (bottom) for ubiquitination to the SCF complex.

differ by only a single amino acid,
whereas RUB3 is ~77% identical to the
other two proteins18. In an in vitro activation assay, the AXR1–ECR1 enzyme
can form a thiolester adduct with all
three RUB proteins but not ubiquitin.
Like ubiquitin activation, RUB activation
is a two-step process. The first step

134

is the formation of adenylated RUB.
Analysis of RUB1 activation has revealed that the ECR1 protein is sufficient to generate AMP–RUB1, but AXR1
is required for the formation of the
RUB1–ECR1 thiolester adduct19.
Once activated, RUB1 is transesterified to the RUB1-conjugating enzyme
(RCE1)19. In yeast and mammals, RUB1
(NEDD8 in mammals) is conjugated to
members of the cullin protein family20,21. Cullins are components of a variety of multisubunit E3 ubiquitin ligases,
including SCF complexes (for Skp1p,
Cdc53p/cullin and F-box protein),
CBCVHL (for cullin, elonginB/C and the
von-Hippel-Lindau tumor suppressor
protein) and the APC (anaphase-promoting complex). An Arabidopsis cullin
called AtCUL1 is also modified by
RUB119,22 (Fig. 1b). When expressed and
purified from a reticulocyte lysate,
AtCUL1 can be RUB1-modified in a reaction mixture containing recombinant

AXR1, ECR1, RCE1, RUB1 and ATP. RUB1
cannot be conjugated to a mutant
derivative of AtCUL1 containing a
K722M substitution. This residue is conserved in all cullins and is likely to be
the site of RUB1 attachment. These results suggest that at least in vitro RUB1
conjugation does not require E3 activity. Alternatively, it is possible that
factors derived from the reticulocyte

lysate copurify with AtCUL1 and provide E3 activity in the conjugation
assay.
Genetic evidence in yeast suggests
that Rub1p modification of the Cdc53p
cullin affects the function of some SCF
ubiquitin ligases20. The SCFs define a superfamily of E3s that employ combinatorial control to mediate ubiquitin-ligase
specificity. The core of the complex
consists of Skp1p, Cdc53p, the recently
identified Rbx1p/Roc1p/Hrt1p and the
ubiquitin-conjugating enzyme Cdc34p
(Fig. 2). F-box proteins bind to this core
through an interaction between the
F-box domain and Skp1p. The F-box protein acts as an adaptor subunit that
recruits specific substrates to the complex, targeting them for ubiquitination
by Cdc34p. In S. cerevisiae, SCFCdc4 is required for ubiquitination and degradation of the cyclin-dependent kinase
(CDK) inhibitor Sic1p. Although mutations in the yeast Rub1p conjugation
pathway confer no obvious phenotype, a
synthetic lethal phenotype is observed
with double mutants between genes in
the pathway and conditional alleles of
components of SCFCdc4. This suggests
that Rub1p conjugation to Cdc53p has a
role in SCFCdc4 function. Possible functions include facilitating SCF complex
assembly, improving SCF catalytic
efficiency or altering substrate specificity. Unlike mutations in the yeast Rub1
pathway, mutations in the Arabidopsis
RUB conjugation pathway have quite
dramatic consequences, suggesting that
the RUB pathway has a much more
prominent role in higher eukaryotes.
Molecular characterization of the
TIR1 (for auxin transport inhibitor response 1) auxin-response gene has
greatly strengthened the hypothesis
that RUB1 conjugation regulates some
aspect of SCF function. Mutations in
TIR1 confer an auxin-resistance phenotype that is similar to that caused by
mutations in AXR1, although less severe. TIR1 encodes an F-box protein
with a series of leucine-rich repeats15.
Recent findings have confirmed that
TIR1 interacts with plant orthologs of
Skp1p (called ASK1 and ASK2) and a
cullin protein to form SCFTIR1 (Ref. 22).
The cullin in SCFTIR1 is the same AtCUL1
protein that is a substrate for AXR1dependent RUB1 modification. This result is consistent with earlier genetic
studies, which suggested that TIR1 and
AXR1 function in a common pathway15.
In addition, TIR1 and AXR1 exhibit overlapping expression patterns, with highest
levels in tissues actively undergoing cell

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TIBS 25 – MARCH 2000
division or cell expansion17,22 (J.C. del
Pozo and M. Estelle, unpublished).
Additional evidence implicating
SCFTIR1 in auxin response has been obtained by analysing ask1-1 mutants and
transgenic lines that overexpress TIR1.
The ask1-1 mutation was identified in a
screen for male sterile plants23. Mutant
plants also exhibit several phenotypes
consistent with reduced auxin response,
including decreased lateral root development and resistance to applied
auxin22. ASK1 is a member of a family of
Skp1-like proteins in Arabidopsis that
includes at least ten members. The isolation of ask1 mutants, along with the
discovery that TIR1 does not interact
with all of the Skp1-like proteins in a twohybrid assay22, implies that individual
family members have distinct functions.
This could provide an additional layer of
SCF combinatorial control in higher
eukaryotes.

(a)

Aux/IAA
I

(b)

II

III

IV

III

IV

ARF
DNA binding

MR

The auxin-regulated genes
The Aux/IAA genes are rapidly (10–60
minutes) and specifically induced by
auxin24. There are at least 20 members of the Aux/IAA gene family in
Arabidopsis, and many members display
distinct basal and induced expression
levels, induction kinetics and patterns
of expression. Because cycloheximide
treatment also induces many of these
genes, it has been proposed that a shortlived repressor could negatively regulate
their transcription. The Aux/IAA proteins are 20–35 kDa and share four
highly conserved domains (Fig. 3a).
Domain III is related to the baa-fold in
b-ribbon DNA-binding domains of the
Arc and MetJ prokaryotic transcriptional
repressor proteins, suggesting that
these factors might function as transcriptional regulators. Consistent with
this possibility, several of these proteins
have been localized to the nucleus.
Several members of the Aux/IAA family are capable of forming homo- and
heterodimers through domains III and
IV25. Additionally, some can interact
with members of a second protein family, including the DNA-binding protein
auxin-response factor 1 (ARF1)26,27.
Members of the ARF protein family
share domains III and IV with the
Aux/IAA proteins (Fig. 3a). The ARFs
bind to a 6-bp auxin-response element
(AuxRE) found upstream of several
auxin-inducible genes and apparently
function to regulate transcription of
these genes28,29. Ulmasov et al.27 have
demonstrated by cotransfection of carrot protoplasts that some members of

Ti BS

Figure 3
The Aux/IAA (auxin/indole-3-acetic acid-induced) and ARF (auxin-responsive factor) protein
families. (a) Schematic diagram of the domain organization of the Aux/IAA and ARF protein
families. Canonical Aux/IAA proteins share four highly conserved domains. Ten members of
the ARF protein family have been identified, all of which contain the Aux/IAA domains III and
IV, except for ARF3/ettin28. Domains III and IV mediate dimerization. The middle region
(MR) of several of the ARF proteins contain biased amino acid sequences (e.g glutaminerich) that have been proposed to function as transcriptional activation or repression domains26. (b) Wild-type (wt), axr3-1 and axr3-2 plants. The gain-of-function axr3 mutations
confer increased auxin response. For example, compare the increased apical dominance
(the inhibition of lateral branching) of the axr3 mutants with the reduced apical dominance
of axr1-12 mutant plants (Fig. 1a). Bar, 5 cm.

the ARF family activate transcription of
auxin-inducible reporters, whereas others repress expression of these reporter
genes. Furthermore, overexpression of
some Aux/IAA proteins represses transcription of an AuxRE-reporter26.
Because the Aux/IAA proteins are not
believed to bind directly to AuxREs,
this negative regulation could occur
through interaction with ARFs. Taken
together, these results illustrate the
complexity of auxin-mediated gene
regulation. Literally hundreds of different Aux/IAA–Aux/IAA, ARF–ARF and Aux/
IAA–ARF combinations could modulate
auxin-regulated gene expression in response to changes in auxin concentration
and other developmental cues.
Recent genetic evidence has solidified
the importance of the Aux/IAA gene family
in auxin response. Mutations in the AXR3
gene were isolated in a screen for
seedlings resistant to low concentrations

of applied auxin. The semi-dominant axr3
mutants exhibit elevated auxin responses,
including increased apical dominance, adventitious rooting and ectopic auxininducible gene expression30 (Fig. 3b).
Cloning of AXR3 revealed that it encodes
the Aux/IAA family member IAA17
(Ref. 31). Dominant mutations in the SHY2
and AXR2 genes also confer auxin-related
growth defects. SHY2 and AXR2 were recently cloned and found to encode
Aux/IAA proteins32 (P. Nagpul, L. Walker,
M. Estelle, J. Reed, unpublished).
Curiously, mutations in AXR3, SHY2 and
AXR2 all occur within a small, highly conserved region of domain II. Analysis of intragenic revertants of AXR3 and SHY2 indicates that the original mutations in these
genes confer a gain of function. This could
be a dominant-negative effect resulting
from the formation of non-functional
dimers with ARF or Aux/IAA proteins. An
alternative explanation favored by these

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Regulation of RUB1
conjugation?

Auxin

Auxin-dependent
kinase?

E1
RCE1

E2

AS
K
TI 1
R1

AtCul1

Rbx1

ECR1
AXR1

Repressor

Repressor

ss
or

SCFTIR1 complex

RUB1

R

ep

re

Ubiquitin

Early-auxinresponse genes

Proteasome

Auxin-mediated
growth and development

Ti BS

Figure 4
A model for auxin response. In response to auxin, SCFTIR1 (for Skp1p, Cdc53p/cullin and
F-box protein, with TIR1 as the F-box protein), functions as a ubiquitin ligase that targets
negative regulators of the auxin-response pathway for ubiquitin-mediated degradation.
AXR1–ECR1-dependent RUB1 modification of the AtCUL1 protein is required for SCFTIR1
function. Degradation of the negative regulator(s) derepresses the auxin-response pathway,
resulting in the transcriptional activation of early-response genes (e.g. the Aux/IAA genes),
which might regulate downstream genes involved in auxin-mediated growth and development. How auxin regulates this pathway is unclear. Two possibilities are the regulation of
RUB1 conjugation (left) and the activation of a protein kinase that phosphorylates the
putative repressor protein, promoting association with SCFTIR1 (right). Abbreviations: AXR1,
auxin resistant 1; ECR1, E1 C-terminus-related 1; RCE1, RUB1-conjugating enzyme 1;
RUB1, related to ubiquitin 1.

authors is that the mutant proteins exhibit
increased stability. Several wild-type
Aux/IAA proteins have been shown to be
short-lived with half-lives of 6–8 minutes33.
Thus, dominant mutations that stabilize
these proteins could affect downstream
gene expression, thus altering auxin
response.

A model for auxin response
Figure 4 presents one possible model
for the AXR1–TIR1 pathway in auxin signaling. In response to hormone, TIR1 recruits one or more repressors of auxin
response to an SCF ubiquitin-ligase complex. Ubiquitination of this repressor is
dependent upon the AXR1–ECR1-mediated RUB1 modification of AtCUL1.
Subsequent degradation of the repressor via the 26S proteasome derepresses
the auxin-response pathway resulting
in the expression of auxin-regulated

136

genes and auxin-mediated growth and
development.
The putative repressor targeted for
degradation by SCFTIR1 is unknown. One
possible candidate is the SAR1 (for suppressor of auxin resistance 1) gene
product. Recessive mutations in SAR1
were isolated in a screen for suppressors of axr1 (Ref. 34). In addition to suppressing nearly every aspect of the axr1
phenotype, sar1 mutations also confer a
distinct phenotype that is epistatic to
axr1, suggesting that SAR1 acts downstream of AXR1 in the auxin-response
pathway. Given that loss of SAR1 largely
alleviates the need for AXR1 in auxin response, one attractive possibility is that
SAR1 is the target of the AXR1–TIR1
pathway. This seems somewhat unlikely,
however, given that sar1 mutants do not
display any constitutive auxin-response
phenotypes, and mutations in sar1 do

not suppress mutations in tir1 (A.
Cernac and M. Estelle, unpublished).
Thus, SAR1 could act at a point between
AXR1 and TIR1. One possibility is that
SAR1 encodes a protein that removes
RUB modifiers from their substrates. Li
and Hochstrasser35 recently identified a
yeast protease that is required for cellcycle progression. This protease specifically cleaves the Smt3p/SUMO-1 Ubl
from substrates, thus demonstrating
that Ubl removal is an important cellular
function.
Members of the Aux/IAA family are also
potential substrates of SCFTIR1. As discussed above, these proteins have short
half-lives, suggesting that they might
be regulated by ubiquitin-mediated
degradation. Furthermore, certain members of this family negatively
regulate auxin-inducible gene expression.
Thus, it seems possible that a regulatory loop exists where the basal expression of specific Aux/IAA proteins represses the auxin response pathway,
and auxin relieves inhibition by promoting the ubiquitin-mediated degradation
of these factors. Removal of the repressors causes a transient auxin response,
including the increased expression of
Aux/IAA genes, which downregulate the
response pathway (Fig. 5). The direct
measurement of Aux/IAA protein stability in axr1 and tir1 mutants is an important test of this model.

Where is the regulation?
Despite these advances in our understanding of auxin action, the mechanisms of auxin perception and regulation of the AXR1–TIR1 pathway are
still largely a mystery. Although several
auxin-binding proteins have been identified, convincing evidence for receptor function has remained elusive.
Nonetheless, recent findings with the
Arabidopsis auxin-binding protein 1
(ABP1) are encouraging. Using an inducible expression system, Alan Jones
and colleagues have demonstrated that
ABP1 promotes auxin-dependent cell expansion when overexpressed in tobacco
and maize leaf cells36. It will be interesting to determine whether this effect is
dependent on the AXR1–TIR1 pathway.
If ABP1 does indeed function as an
auxin receptor in the AXR1–TIR1 pathway, it is still unclear how the signal is
transduced. The sequence of ABP1 resembles no other proteins in the data
base, thus providing no information on
what types of signaling mechanism might
be employed. The identification of an SCF
complex in the auxin response suggests

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complexity observed in the developing
plant.

Acknowledgements

Aux/IAA

The research in the authors’ laboratory is supported by NIH GM 43644 and
DOE DE-FG02-98-ER20313 to M.E. and
NIH GM 18680 to W.M.G.

Aux/IAA

References

+ auxin

SCFTIR1

Aux/IAA

Aux/IAA

Downstream genes
SCFTIR1
Ti BS

Figure 5
The Aux/IAA (auxin/indole-3-acetic acid-induced) proteins as substrates of the AXR1–TIR1
pathway. In this model, some members of the Aux/IAA family function as repressors (red),
whereas others are positive regulators of auxin response (blue). For example, phenotypic
analysis of axr3 mutants suggests that AXR3/IAA17 positively regulates auxin response,
whereas other IAA proteins negatively control auxin response. Basal levels of repressor
Aux/IAA proteins repress expression of all Aux/IAA genes. Auxin promotes the ubiquitinmediated degradation of the repressors via SCFTIR1, resulting in the derepression of
Aux/IAA transcription. Newly synthesized, positively acting Aux/IAA proteins regulate the
expression of downstream genes involved in auxin response. In addition, the accumulation
of newly synthesized negatively acting Aux/IAA proteins restores repression. Abbreviations:
SCFTIR1, Skp1p, Cdc53p/cullin and F-box protein, with TIR1 as the F-box protein.

that phosphorylation-based signaling
pathways might be involved. All known
substrates of SCF ubiquitin ligases must
be phosphorylated to trigger their association with the SCF (Refs 5,37). Perhaps, an
auxin-activated kinase phosphorylates
substrates of SCFTIR1. Auxin-stimulated
kinases have been identified38, but genetic evidence implicating them in auxin
response is lacking.
Another possible avenue for auxin
input is through regulation of E3 activity. Auxin could potentially regulate the
assembly, localization, specificity or activity of SCFTIR1. One possible means by
which this could occur is by regulated
RUB1 modification of AtCUL1.

Concluding remarks
The complexity of auxin physiology
has been appreciated for many years.

Auxin is involved in virtually every aspect
of plant growth and development, evokes
a diversity of rapid biochemical changes,
and has disparate effects on cell growth,
depending on the context. It should therefore come as no surprise that the machinery of auxin signal transduction and
auxin response is also complex. In addition to the large Aux/IAA and ARF families
of transcriptional regulators, there are
likely to be multiple SCFs that function in
auxin response. So far, three close relatives of TIR1, called Leucine-Rich Repeat Fbox (LRF), have been identified in the
Arabidopsis genome. The proteins encoded by these genes are likely to interact with specific members of the ASK and
cullin protein families to form specialized
SCFs. A major challenge for future studies
is to determine how this molecular
complexity relates to the physiological

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Structural comparisons

A novel FeS cluster in Fe-only
hydrogenases
Yvain Nicolet, Brian J. Lemon, Juan C.
Fontecilla-Camps and John W. Peters
Many microorganisms can use molecular hydrogen as a source of electrons
or generate it by reducing protons. These reactions are catalysed
by metalloenzymes of two types: NiFe and Fe-only hydrogenases. Here, we
review recent structural results concerning the latter, putting special
emphasis on the characteristics of the active site.
IN 1931, Stephenson and Stickland
showed that the facultative anaerobic
colon bacterium Escherichia coli could activate hydrogen thanks to enzymes they
termed hydrogenases1. More recently,
these enzymes have been shown to play a
central role in the hydrogen metabolism
of many microorganisms of great biotechnological interest, such as methanogenic,
acetogenic, nitrogen-fixing, photosynthetic and sulfate-reducing bacteria.
Knowledge of the functional and
structural properties of hydrogenases
might help in the design of less expensive catalysts for fuel cells. Existing fuel
cells use platinum catalysts and these
severely restrict the use of hydrogendriven vehicles in spite of the urgent
need to switching to clean fuels2.
Hydrogenases catalyse the reversible reaction H2 ↔ 2H1 1 2e2 (Refs 3–6). The first
Y. Nicolet and J.C. Fontecilla-Camps are at
the Laboratoire de Cristallographie et de
Cristallogenèse des Protéines, Institut de
Biologie Structurale Jean-Pierre Ebel, CEACNRS, 41 Avenue des Martyrs, 38027,
Grenoble Cedex 1, France; and B.J. Lemon
and J.W. Peters are at the Dept of Chemistry
and Biochemistry, Utah State University,
Logan, UT 84322-0300, USA.
Email: juan@lccp.ibs.fr; petersj@cc.usu.edu

138

step in the reaction is the heterolytic cleavage of H2 into H1 and H2. The hydrogenuptake reaction results in protons and
electrons, which are subsequently used
to generate ATP and reducing power;
the hydrogen-producing reaction involves the reduction of protons by lowredox-potential electrons generated by
fermentation. Most of the hydrogenases
described to date are metalloproteins
containing Ni or Fe, or both. Of these, the
NiFe hydrogenases are the most extensively studied and the three-dimensional
structures of several enzymes belonging
to this group have been reported7. A surprising feature of these enzymes is that
their active sites contain, in addition to a
Ni ion, a redox-inactive, low-spin Fe21 center with CO and CN2 coordination8,9.
The unrelated Fe-only hydrogenases
have been less well studied and, until
very recently, no three-dimensional
structure was available for this class of
enzyme. However, in the past year or so,
the structures of the Fe-only hydrogenases from Desulfovibrio desulfuricans
and Clostridium pasteurianum have been
solved to 1.6 and 1.8 Å resolution, respectively10,11. Here, we compare these
structures and discuss their analogies
to the NiFe-containing enzymes.

Both amino acid sequence analyses
and electronic paramagnetic resonance
(EPR) studies have indicated that Feonly hydrogenases generally contain
two [4Fe–4S] clusters (the F-clusters) in
a ferredoxin-like domain. In addition, an
unusual EPR signal has been attributed
to a novel 6Fe cluster that was proposed
to be the active site and was called the
H-cluster5. The positions of the F- and Hclusters in the respective F- and Hdomains of C. pasteurianum hydrogenase I
(CpI) and D. desulfuricans hydrogenase
(DdH) are shown in Fig. 1. DdH is a
dimeric periplasmic protein of 53 kDa
(43 kDa and 10 kDa for the large and
small subunits, respectively) and CpI is a
monomeric cytoplasmic enzyme of 61 kDa.
In addition to the clusters described
above, CpI contains two FeS centers coordinated by domains found at the N
terminus of the molecule. One of these
domains bears a striking structural similarity to [2Fe–2S] plant-type ferrodoxins
(green in Fig. 1b,c). A short domain (pink
in Fig. 1b,c) connects the N-terminal
[2Fe–2S] ferrodoxin-like module with
the F-domain and consists of just two a
helices separated by a loop region that
coordinates an additional [4Fe–4S] cluster through three cysteinyl ligands and
the Ne ring atom of a histidine residue.
As was expected from the high degree
of amino acid sequence identity
(Fig. 1a)12,13, CpI and DdH display extensive structural similarities at the Hand F-domains (Fig. 1b,c)10,11. The rootmean-square deviation (rmsd) for the
superposition between the H-domains is
1.0 Å (338 Ca’s) and that between the Fdomains is 1.7 Å (57 Ca’s). However, the
relative orientations of the H- and Fdomains are not the same in the two
enzymes: the rmsd for the combined Hand F-domains (381 Ca’s) is 2.2 Å. This is
probably a consequence of differences
elsewhere in the molecules, such as the
two extra N-terminal domains in CpI, the
numerous insertions in the F-domain in
CpI and the longer C-terminal region of

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