UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4°C. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known fivecoordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (Fe III PPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of Fe III PPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.Heme proteins with protein-derived oxygen donor proximal ligands are relatively rare in nature. The best known example of such ligation is that of bovine liver catalase which contains a tyrosine phenolate proximal heme iron ligand (1).In addition, a series of naturally occurring hemoglobin mutants having distal or proximal histidines replaced by tyrosine or glutamate (the M hemoglobins) have been established by X-ray crystallography (2-4) and resonance Raman spectroscopy (5, 6) to have phenolate or carboxylate ligation. Other proteins with tyrosinate and glutamate oxygen donor ligation have been recently produced by site-directed mutagenesis of various myoglobins (7-10) and cytochrome c peroxidase (11).The use of site-directed mutagenesis techniques has become invaluable in identifying catalytically and structurally important protein residues within a protein system. A relatively new type of mutation, based on altering the size of the amino acid at the point of mutation, substitutes a glycine or alanine for the larger original residue and leaves a cavity within the protein. Termed cavity mutants, these mutated proteins demonstrate the ability to employ exogenous ligands to reconstitute their wild-type activity. This rescue of activity has been seen for the cavity mutants of azurin (12), carbonic anhydrase (13), hexose-1-phosphate uridyltransferase (14), and various heme proteins (15-18).

To survive in a dynamic and unpredictable environment, cells must correctly interpret and integrate extracellular signals with internal factors. In particular, internal stores of nutrients must be managed for use during periods of nutrient limitation. To gain insight into this complex process, we combined biochemical and spectroscopic techniques to follow the dynamics of the phosphate responsive signaling pathway in both single yeast cells and populations. We demonstrate that the phosphate-responsive genes PHO5 and PHO84 exhibit different kinetics of transcriptional induction in response to phosphate starvation, and that transient phosphate limitation causes induction of PHO84 but not PHO5. This differential kinetic behavior is largely eliminated in cells that lack the ability to store phosphate internally in the form of polyphosphate, but the threshold of external phosphate required for induction of PHO5 and PHO84 is unaffected. Our observations indicate that polyphosphate acts as a buffer that can be mobilized during periods of phosphate limitation and enables the phosphate-responsive signaling pathway to filter transient fluctuations in extracellular phosphate levels.homeostasis ͉ nutrients ͉ polyphosphate ͉ signaling C ellular responses to environmental cues involve a complex interplay between intracellular conditions and extracellular factors, coordinated via elaborate signal transduction cascades that initiate changes in metabolism and gene regulation. Insight into how cellular responses facilitate survival in dynamic environments can be gained by following the response to deprivation of the essential nutrient, phosphate, in the model organism, Saccharomyces cerevisiae.In response to phosphate starvation, budding yeast cells have three major responses: they mobilize internal stores of phosphate (1, 2), up-regulate production of a plasma membrane transporter involved in phosphate uptake (Pho84), and increase production of phosphatases (e.g., Pho5) that are secreted into the extracellular environment to liberate inorganic phosphate (P i ) by hydrolysis of organic phosphates (3, 4). These responses are mediated primarily at the level of gene regulation and are controlled by a signaling pathway known as the phosphateresponsive signaling (PHO) pathway (5-7). Key components in the PHO signaling pathway include a cyclin͞cyclin-dependent kinase complex, Pho80͞Pho85, whose activity is regulated in response to phosphate conditions, and its transcription factor substrate, Pho4, which regulates phosphate-responsive gene transcription (5,8,9). Phosphorylation of Pho4 on four serine residues by Pho80͞Pho85 controls Pho4 subcellular localization and its interaction with another transcription factor, Pho2 (10).When cells are grown in medium containing a high concentration of P i , Pho80͞Pho85 is active, Pho4 is fully phosphorylated and localized to the cytoplasm, and transcription of phosphateresponsive genes such as PHO5 and PHO84 is turned off (8, 9). In response to phosphate limitation, Pho80͞Pho85 is inactivated, and ...