News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000
Principles of Protein Structure for Chemical Recognition, Complementarity,
E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau, R.M. Brudler, J.M. Castagnetto,
M. DiDonato, E.D. Garcin, U.K. Genick, K.B. Gerwert,* C.J. Kassmann, C.K. Koike,
S.J. Lloyd, T.P.K. Lo, J.-L. Pellequer, M.E. Pique, R.J. Rosenfeld, D.S. Shin,
M.E. Stroupe, M.M. Thayer, M.J. Thompson, J.L. Tubbs, T.T. Woo* Ruhr-Universität,
Biocatalysis of Sulfur Transformations
Sulfite and nitrite reductases catalyze fundamental chemical transformations
for biogeochemical cycling of sulfur and nitrogen. Sulfite reductase catalyzes
the concerted 6-electron reductions of sulfite to sulfide and nitrite to ammonia.
We used multiwavelength anomalous diffraction of the native siroheme, an iron-containing
macrocycle of the isobacteriochlorin class, and Fe4S4 cluster
cofactors to solve the atomic-resolution structure of the hemoprotein from sulfite
We determined 12 high-resolution structures of sulfite reductase hemoprotein
that characterize its active center in 3 different states of oxidation and its
interactions with substrates, inhibitors, intermediates, and products. Coupled
crystallographic and spectroscopic studies revealed the mechanism for heme activation
via reduction-gated exogenous ligand exchange and provided descriptions of the
intermediates at each step along the complex reaction pathway.
We are continuing our exploration of the mechanism by using rational mutagenesis
of the hemoprotein to probe the nature of the interactions between the unique
metal centers and the protein environment. We are also extending the target of
our structural investigation to include the enzyme that prepares the siroheme
for incorporation into sulfite reductase hemoprotein. This enzyme, siroheme synthase,
is required for expression of active sulfite reductase. Determining the structure
of the synthase will provide further insight into the principles that define
interactions between a protein and its cofactors.
Oxygen Recognition and Reactive Oxygen Catalysis
Ongoing research in the Getzoff and Tainer laboratories aims to understand
the unique structural metallobiochemistry of nitric oxide synthases (NOSs) and
superoxide dismutases (SODs), which produce and regulate reactive oxygen species.
Cu,Zn SOD, which dismutes superoxide anions into oxygen and hydrogen peroxide,
is an antioxidant enzyme and a master eukaryotic regulator of reactive oxygen
species. Moreover, by controlling levels of superoxide available for rapid reaction
with nitric oxide to form peroxynitrite, SODs also regulate nitric oxide, an
important biological signal and defensive cytotoxin (see following).
Biochemically, eukaryotic Cu,Zn SODs are remarkable for their unusually great
stability, faster-than-diffusion attraction of substrate, exquisite specificity,
and efficient catalysis. Biologically, SODs are important for their antioxidant,
anti-inflammatory, and antiaging properties. Medically, genetic mutations in
human Cu,Zn SOD cause the fatal degenerative disease of motor neurons termed
amyotrophic lateral sclerosis or Lou Gehrig disease.
Our new structures of bacterial Cu,Zn SODs from symbionts and pathogens provide
the potential for drug design. Whereas the fold and active-site geometry of bacterial
Cu,Zn SODs match those of eukaryotic SODs, the elements recruited to form the
dimer interface and the active-site channel are strikingly different (Fig. 1).
With our structures and redesign of SODs, we aim to elucidate the structural
metallobiochemistry and structure-function relationships of the enzymes.
Our new research on the structural and chemical biology of NOSs, which regulate
the synthesis and thereby the biological activity of nitric oxide, complements
our studies on SODs. Nitric oxide functions at low concentrations as a diffusible,
biological messenger for neurotransmission, long-term potentiation, platelet
aggregation, and regulation of blood pressure. At higher concentrations, nitric
oxide acts as a cytotoxic agent for defense against tumor cells and intracellular
Each NOS subunit is divided into 2 domains joined by a calmodulin-binding
hinge region: (1) an oxygenase domain, with binding sites for heme, tetrahydrobiopterin,
and substrate, that forms the catalytic center for production of nitric oxide
and (2) a reductase domain, with binding sites for NADPH, FAD, and FMN, that
supplies electrons to the heme. We crystallized both domains and determined refined
structures for the oxygenase domain in monomeric and dimeric forms and in complex
with cofactors, cofactor analogs, substrate, intermediate, and inhibitors.
The structures revealed a novel protein fold and characterized the roles
of the cofactors and of the l-arginine substrate itself in the 2 steps of the
reaction mechanism. First, l-arginine is oxidized to N-hydroxy-l-arginine
by a monooxygenase-like reaction; then this intermediate is oxidized to form
citrulline and nitric oxide by an unprecedented reaction. Ongoing structural
and computational studies of NOS-inhibitor complexes are providing an interactive
design cycle for the structure-based design of optimized inhibitors.
Chemical Biology of Per-Arnt-Sim Domains of Sensors and Clock Proteins
We aim to characterize the structural and chemical biology of PER-ARNT-SIM
(PAS) domains. These domains were originally identified as sequence repeats in
the Drosophila clock protein PER and the basic-helix-loop-helix-containing
transcription factors aryl hydrocarbon receptor nuclear translocator (ARNT) in
mammals and single-minded (SIM) in flies. PAS domain sequences, which mediate
protein-protein interaction and in some cases bind small ligands, also occur
in sensor kinase proteins, the phytochrome photoreceptors of plants, and the
newly discovered eukaryotic clock proteins, including the clock proteins that
clearly couple photoreception to circadian rhythms.
The structures of PAS domains resemble the structure of photoactive yellow
protein (Fig. 2), a bacterial blue-light photosensor whose structural molecular
biology has been under study. Two recently determined structures of PAS domains
from the oxygen-sensing heme domain of the bacterial protein FixL and from the
amino-terminal regulatory domain of the voltage-dependent potassium channel HERG
confirmed that photoactive yellow protein is an appropriate structural prototype
of the PAS domains. The aim of research funded by the Skaggs Institute is to
use our 0.83-Å resolution structure of photoactive yellow protein to determine
structures for other PAS domains and to characterize the roles of these domains
in signal transduction. These results provide the basis to ultimately control
PAS domain sensors in plants and animals for numerous applications in biotechnology.
Coupled Crystallographic and Spectroscopic Analysis of Protein Mechanisms
We established a powerful approach to chemical structural biology by developing
state-of-the-art technology that enables us to couple the determination of crystallographic
structure with the chemical insight provided by spectroscopy. Using our laser
and crystal spectroscopy facility, we can initiate reactions in protein crystals,
monitor the reactions on a nanosecond timescale, and characterize transient structural
intermediates along reaction pathways. Using such measurements, we can establish
the conditions required to generate a large proportion of the intermediate in
crystals. We can then apply these conditions to our crystals and by using time-resolved
x-ray crystallography, determine the structure of the intermediate we generated.
By monitoring the spectroscopic signals in protein crystals, we can also precisely
determine the oxidation state of a protein's chromophore or the coordination
environment of its metal center before and after an x-ray crystallographic experiment
Having successfully applied the time-resolved techniques to photocycle intermediates
of photoactive yellow protein, we are extending the technique to probe the mechanism
of other systems, including nitric oxide synthase and sulfite reductase. Nitric
oxide synthase catalyzes the production of the physiologic signaling molecule
nitric oxide. We are using a coupled spectroscopic and crystallographic approach
to generate and study product-inhibited complexes and to follow oxidation states
during the catalytic cycle. Combining techniques will help further our understanding
of catalytic cycles in signal transduction proteins. Recent accomplishments included
defining the structural chemistry of the pterin site.
Sulfite reductase helps metabolize sulfur by reducing sulfite to sulfide.
We are interested in understanding the structural relationship between the protein,
its metal-containing cofactors, and its chemical function. Using our combined
spectroscopic and structural technique, we are elucidating the principles that
govern the unique 6-electron reaction of sulfite reductase.
Adak, S., Crooks, C., Wang, Q., Crane, B.R., Tainer, J.A., Getzoff, E.D.,
Stuehr, D.J. Tryptophan 409 controls the activity of neuronal nitric-oxide
synthase by regulating nitric oxide feedback inhibition. J. Biol. Chem. 274:26907,
Crane, B.R., Arvai, A.S., Ghosh, S., Getzoff, E.D., Stuehr, D.J., Tainer,
J.A. Structures of the Nw-hydroxyl-L-arginine complex of
inducible nitric oxide synthase oxygenase dimer with active and inactive pterins.
Biochemistry 39:4608, 2000.
Crane, B.R., Rosenfeld, R.J., Arvai, A.S., Ghosh, D.K., Ghosh, S., Tainer,
J.A., Stuehr, D.J., Getzoff, E.D. N-terminal domain swapping and metal ion
binding in nitric oxide synthase dimerization. EMBO J. 18:6271, 1999.
Demchuk, E., Genick, U.K., Woo, T.T., Getzoff, E.D., Bashford, D. Protonation
states and pH titration in the photocycle of photoactive yellow protein. Biochemistry
Devanathan, S., Brudler, R., Hessling, B., Woo, T.T., Gerwert, K., Getzoff,
E.D., Cusanovich, M.A., Tollin, G. Dual photoactive species in Glu46Asp and
Glu46Ala mutants of photoactive yellow protein: A pH-driven color transition.
Biochemistry 38:13766, 1999.
Forest, K.T., Langford, P.R., Kroll, J.S., Getzoff, E.D. Cu,Zn superoxide
dismutase structure from a microbial pathogen establishes a class with a conserved
dimer interface. J. Mol. Biol. 296:145, 2000.
Ghosh, D.K., Crane, B.R., Ghosh, S., Wolan, D., Gachhui, R., Crooks, C.,
Presta, A., Tainer, J.A., Getzoff, E.D., Stuehr, D.J. Inducible nitric oxide
synthase: Role of the N-terminal ß-hairpin hook and pterin-binding segment
in dimerization and tetrahydropterin interaction. EMBO J. 18:6260, 1999.