Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

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The hemoglobin of yeast is a two-domain protein with both heme and flavin prosthetic groups. The nucleotide sequences of the cDNA and genomic DNA encoding the protein from Saccharomyces cerevisiae show that introns are absent and that both domains are homologous with a flavoheme protein from Escherichia coli. The heme domains are also homologous with those of 02-binding heme proteins from several other distantly related bacteria, plants, and animals; all appear to be members of the same globin superfamily. Although the homologous hemoglobin of the bacterium Vitreoscilla sp. is a single-domain protein, several bacteria have related 02-binding heme proteins whose second domains have different structures and enzymatic activities: dihydropteridine reductase (E. colb), cytochrome c reductase (Akaligenes eutrophus), and kinase in the 02 sensor of Rhizobium melilod. This indicates that one evolutionary pathway of hemoglobin is that of a multipurpose domain attached to a variety of unrelated proteins to form molecules with different functions. The flavin domain of yeast hemoglobin is homologous with members of a flavoprotein family that includes ferredoxin reductase, nitric oxide synthase, and cytochrome P-450 reductase. The correspondence of yeast and E. coli flavohemoglobins indicates that the two-domain protein has been conserved intact for as long as 1.8 billion years, the estimated time of divergence of prokaryotes and eukaryotes provided that cross-species gene transfer has not occurred.

ERK2 is a proline-directed protein kinase that displays a high specificity for a single threonine (Thr-38) on the substrate Ets-1, which lies within the consensus sequence 36phi-chi-Thr-Pro39 (where phi is typically a small hydrophobic residue and chi appears to be unrestricted). Thr-38 lies in a long flexible N-terminal tail (residues 1-52), which also contains a second potential phosphorylation site, Ser-26. How Ets-1 binds ERK2 to promote the phosphorylation of Thr-38 while simultaneously discriminating against the phosphorylation of Ser-26 is unclear. To delineate the details of the molecular recognition of Ets-1 by ERK2, the binding of various mutants and truncations of Ets-1 were analyzed by fluorescence anisotropy. The data that were obtained support the notion that the N-terminal tail contains a previously unrecognized docking site that promotes the phosphorylation of Thr-38. This new docking site helps assemble the complex of Ets-1 and ERK2 and makes a similar contribution to the stabilization of the complex as does the pointed domain of Ets-1. The in vitro activation of ERK2 by MKK1 induces a large conformational transition of the activation segment (DFG-APE), but neither induces self-association of ERK2 nor destabilizes the stability of the ERK2.Ets-1 complex. This latter observation suggests that interactions intrinsic to the active site are not important for complex assembly, a notion further supported by the observation that the substitution of a number of different amino acids for Pro-39 does not destabilize the complex. Mutagenesis of ERK2 within loop 13 suggests that Ets-1 binds the substrate-binding groove. These data suggest that ERK2 uses two weak docking interactions to specifically assemble the complex, perhaps in doing so denying Ser-26 access to the active site. Displacement of residues 1-138 of Ets-1 (EtsDelta138) from ERK2 by the peptide N-QKGKPRDLELPLSPSL-C, derived from Elk-1, suggests that Ets-1 engages the D-recruitment site (beta7-beta8 reverse turn and the alphaD-alphaE helix) of ERK2. Displacement of EtsDelta138 from ERK2 by the peptide N-AKLSFQFPS-C derived from Elk-1 shows that EtsDelta138 communicates with the F-recruitment site of ERK2 also.

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O 2 (K I (CO) ‫؍‬ ϳ1 M). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O 2 from inhibitory NO and CO.Investigations of NO toxicity and defenses in Escherichia coli led to the isolation of an NO-inducible NAD(P)H, O 2 , and FAD-dependent NOD 1 activity. This enzyme activity was identified with the flavoHb encoded by the hmp gene (1, 2). More recently, we have shown that the flavoHb efficiently converts NO and O 2 to nitrate via a conventional two electron flavoenzyme mechanism (3). The proposed NOD reaction stoichiometry is described by Equation 1.A function for flavoHbs in NO dioxygenation and detoxification is supported by a growing body of evidence. FlavoHbs are induced by NO, nitrite, nitrate, and NO-releasing agents in various bacteria (2, 4 -9), and these flavoHbs protect bacteria, yeast, and Dictyostelium discoideum against growth inhibition and NO-mediated damage during exposures to authentic NO or NO releasing agents (1, 2, 6, 7, 10 -12). Further, O 2 is required for the robust NO scavenging action of flavoHb both in E. coli and in vitro, and O 2 is required for the maximal protection of cells and NO-sensitive aconitases from NO-mediated damage (1, 2). Other flavoHb mechanisms may also protect cells against nitrosothiol or NO toxicity in the absence of O 2 . FlavoHbs may reduce NO or denitrosylate toxic nitrosothiols formed from NO, sequester NO or reactive heme, or catalyze other beneficial reactions (6,10,13,14). These additional flavoHb activities and functions require consideration.We have now investigated the NOD activities of flavoHbs isolated from Saccharomyces cerevisiae, Alcaligenes eutrophus, and E. coli and have compared these activities with other enzymic activities of flavoHbs including NO reductase, NADH oxidase, and FAD reductase activities. Spectra for the various O 2 , NO, and CO liganded flavoHbs and the transient kinetics for O 2 , NO, and CO binding are reported for flavoHbs and are discussed in relation to the susceptibility of NOD activity to NO and CO inhibition. The kinetics and stoichiometry of the NOD activity indicate a highly specific and efficient dioxygenase mechanism and function for the flavoHbs. MATERIALS AND METHODSReagents-NADPH, FAD, phenylmethylsulfonyl fluoride, and Aspergillus niger glucose oxidase (225 units/mg) were purchased from Sigma. NADH, Aspergillus nitrate reductase, and bovine liver catalase