To understand how a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a bacterial blue-light photosensor. Sequence hom ologies suggest that the PYP fold is the structural prototype for the superfamily of PAS domains found in diverse biological sensors and clock proteins. We are extending our dark-state PYP structure to 0.83-Å resolution, where individual atoms appear as s pheres (Fig. 1). We used millisecond time-resolved Laue crystallography and simultaneous optical spectroscopy to determine the first atomic structure for a protein photocycle intermediate. The two PYP structures reveal the synergistic interactions between the chromophore and the protein that tune the spectral and kinetic properties of the light cycle for efficient protein-mediated signal transduction. We made PYP proteins with site-directed mutations and modified chromophores by recombinant expression an d chemical attachment of the chromophore to experimentally test hypotheses for the light-cycle mechanism.

Sulfite and nitrite reductases catalyze fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen. We determined the 1.6-Å crystallographic structure of sulfite reductase hemoprotein, which catalyzes the concerted six-electron reductions of sulfite to sulfide and nitrite to ammonia, by using multiwavelength anomalous diffraction (MAD) of the native siroheme and Fe₄S₄ cluster cofactors, multiple isomorphous replacement, and selenomethionine sequence marker s. A distinctive three-domain a/b fold controls cofactor assembly and reactivity, and identifies a sulfite or nitrite reductase repeat common to a redox-enzyme superfamily. Coupled spectroscopy and crystal lography of the enzyme in three oxidation states showed that heme activation occurs via reduction-mediated ligand exchange. Refined crystallographic complexes with substrates, inhibitors, intermediates, and products demonstrated how the active site facili tates the reaction and accommodates the varied reaction intermediates without release (Fig. 2).

The fish Root-effect hemoglobins act as acid-controlled molecular oxygen pumps delivering oxygen against high oxygen pressures to the swim bladder for neutral buoyancy and to the retina for visual acuity. Our 2-Å crystal structures of the ligand-bound hem oglobin from the fish Leiostomus xanthurus show that key Root effect residues recruit conserved residues to strategically assemble positive-charge clusters that promote the extremely pH-dependent allosteric R--T switch with concomitant release of oxygen .

For SOD metalloenzymes, we solved structures of bacterial, bovine, and human mutant enzymes to characterize the structural basis for their activity and stability. Structures of the oxi dized and reduced states of bovine SOD and active-site mutants of human SOD provide information on the enzyme's mechanism. Our bacterial SOD structure is representative of the class of SODs from bacterial pathogens and provides the potential for drug desi gn. Whereas the bacterial SOD subunit fold and active-site geometry match human SOD, the elements recruited to form the dimer interface, the active-site channel and the disulfide bond, are strikingly different. Our SOD structural results suggest a hypothe sis for the mechanism by which single-site mutations in human SOD cause the fatal degenerative disease amyotrophic lateral sclerosis (ALS).

Antibody interactions are a final focus for characterizing protein recognition, function, and interaction. Cytochrome c-bound and free structures of the E8 antibody provided information on conformational changes upon binding, electrostatic interact ions and the key role of water molecules at the antibody-antigen interface. With V. Roberts and S. Benkovic, we designed, constructed, and characterized metalloantibodies and catalytic antibodies that exploit the versatility of the sequence-variable, but structurally conserved antibody scaffold.

Boissinot, M., Karnas, S., Lepock, J.R., Cabelli, D.E., Tainer, J.A., Getzoff, E.D., Hallewell, R.A. Function of the Greek Key Connection Analysed Using Circular Purmutants of Superoxide Dismutase. EMBO J., 16:2171, 1997.

Bourne, Y., Redford, S.M., Steinman, H.M., Lepock, J.R., Tainer, J.A., Getzoff, E.D. Novel dimeric interface and electrostatic recognition. Proc. Natl. Acad. Sci. USA, 93:12774, 1996.

Crane, B., Siegel, L., Getzoff, E.D. Probing the Catalytic Mechanism of Sulfite Reductase by X-ray Crystallography: Structure of the E. coli Hemoprotein in Complex with Substrates, Inhibitors, Intermediates and Products. Biochemistry, accept ed.

Crane, B., Siegel, L., Getzoff, E.D. Structures of the Siroheme and Fe₄S₄-Containing Active Center of Sulfite Reductase in Different States of Oxidation: Heme Activation via Reduction-Gated Exogenous Ligand Exchange. Biochemistry , accepted.

Crane, B.R., Bellamy, H., Getzoff, E.D. Multiwavelength Anomalous Diffraction of Sulfite Reductase Hemoprotein: Making the Most of MAD Data. Acta Crystallographica, D53:8, 1997.

Crane, B.R., Getzoff, E.D. Determining Phases and Anomalous Scattering Models from the Multiwavelength Anomalous Diffraction of Native Protein Metal Clusters. Imporved MAD Phases Error Estimates and Anomalous Scatterer Positions. Acta Crystallographica , D53:23, 1997.

Crane, B.R., Getzoff, E.D. The Relationship Between Structure and Function for the Sulfite Reductases. Current Opinion in Structural Biology, 6:744, 1996.

Devanathan, S., Genick, U.K., Getzoff, E.D., Meyer, T.E., Cusanovich, M.A., Tolin, G. Preparation and Properties of a 3,4-Dihydroxycinnamic Acid Chromophore Variant of the Photoactive Yellow Protein. Archives of Biochemistry and Biophysics, 340:83, 1997.

Fisher, C.L., Cabelli, D.E., Hallewell, R.A., Beroza, P., Lo, T.P., Getzoff, E.D., Tainer, J.A. Computational, Pulse-Radiolytic, and Structural Investigations of Lysine-136 and Its Role in the Electrostatic Triad of Human Cu,Zn Superoxide Dismutase. Pr oteins: Structure, Function and Genetics, in press.

Genick, U.K., Borgstahl, G.E.O., Ng, K., Ren, Z., Pradervand, C., Burke, P.M., Srajer, V., Teng, T.-T., Schildkamp, W., McRee, D.E., Moffat, K., Getzoff, E.D. Structure of a Protein Photocycle Intermediate by Millisecond Time-Resolved Crystallography. Science, 275:1471, 1997.

Genick, U.K., Devanathan, S., Meyer, T.E., Canestrelli, I.L., Williams, E., Cusanovich, M.A., Tollin, G., Getzoff, E.D. Active Site Mutants Implicate Key Residues for Control of Color and Light Cycle Kinetics of Photoactive Yellow Protein. Biochemistry , 36:8, 1997.

Mylvaganam, S.E., Bonaventura, C., Bonaventura, J. Getzoff, E.D. Structural Basis for the Root Effectin Haemoglobin. Nature Structural Biology 3:275, 1996.