News and Publications

Principles of Protein Structure for Chemical Recognition, Complementarity, and Catalysis

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 reductase hemoprotein.

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 parasites.

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 is done.

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.

Publications

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, 1999.

Crane, B.R., Arvai, A.S., Ghosh, S., Getzoff, E.D., Stuehr, D.J., Tainer, J.A. Structures of the N^w-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 39:1100, 2000.

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.