Comparison of resonance Raman spectra of Ddh and Did molecular forms of octaethylporphinatonickel(Il) (NiOEP) indicates high-frequency bands at 1660, 1609, 1581, and 1524 cm-1 are structure-sensitive. Upon deuteration at the meso carbons two additional anomalously polarized bands appear with a concomitant decrease in intensity of the 1310-cm_1 anomalously polarized line. The spectrum in solution is consistent with a planar NiOEP structure. Spectra of CuOEP imply the existence of two molecular forms in this compound. An empirical correlation between the position of the anomalously polarized line at -! 590 cm-1 and the distance from the center of the porphyrin ring to the pyrrole nitrogen is applied to hemeprotein resonance Raman data. It is inferred that out-of-plane displacements of the iron atom are 0.4 Á in deoxyhemoglobin and 0.3 Á in fluoromethemoglobin.were formed18 by addition of excess dithionite to aqueous solutions of the ligated ferric porphyrin. A solution of pyridine protoporphyrin IX iron(II) was prepared at pH 6.8 by solubilizing the porphyrin with 3% sodium dodecylsulfate. FenOEP(L)2, L = imidazole or pyridine, were synthesized by a procedure of Kobayashi.19 CoinOEPX, X-= Bror CIO4-, were obtained by bromine oxidation or electrolysis.20 Samples of the two crystal forms of NiOEP were obtained with the conditions reported by Cullen and Meyer;12-13 however, the preparation of the tetragonal form was contaminated with triclinic crystals. The long needles, characteristic of the triclinic crystal, were removed manually. Triclinic crystals were prepared with hexane-dichloromethane solutions. Densities of the crystals agree with reported values.OctaethyIporphyrin-meso-d4. This compound was prepared21 from 2 -4 by exchange in D2SO4-D2O. After one exchange a minimum of 94% deuterium incorporation at the meso carbons was observed (*H NMR spectrum).Octaethylporphinato -meso-dd -nickel(II). The nickel was inserted in pyridine solvent. Following metal insertion, the solution was added to water and NiOEP extracted into CH2CI2. The solution was washed five times with water, dried over Na2S04, and filtered, and NiOEP crystallized from a CH2Cl2-hexane solution. The crystals were dried in vacuo at 150°. Deuterium incorporation at the meso carbons was 1 (dj), 4 (di), 9 (d3), and 86% (¿4).Chlorooctaethylporphinato-meso-d4-iron(III). Synthesis is the same as for the nickel compound, except DMF was employed as solvent. Deuterium incorporation was 92% (mass spectrum).Nitrosyletioporphyrinatocobalt. NO was bubbled slowly for 10 min into a CHCI3 solution of cobaltous etioporphyrin I. Stirring was continued for an additional 30 min under the NO atmosphere. Argon was bubbled through the solution to remove excess NO, methanol was added, and after 2 days dark purple crystals were formed. The crystals were filtered and washed with methanol: ir (KBr) 1665 cm-1 (NO).

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Activation (phosphorylation) of the transcription factor encoded by spo0A is essential for the initiation of sporulation in Bacillus subtilis. At least three histidine protein kinases are involved in the phosphorylation of Spo0A. Under some growth conditions, KinA was the primary kinase, but under other conditions, KinB had the more critical role. KinC was required for the initial activation of Spo0A, even in the presence of KinA and KinB.Under appropriate conditions, cells of the gram-positive soil bacterium Bacillus subtilis differentiate to form dormant heatresistant endospores. One of the key factors determining whether cells initiate sporulation is the accumulation of the active phosphorylated form of the transcription factor encoded by spo0A (reviewed in reference 7). Spo0A belongs to the response regulator family of transcription factors (1, 20) which usually receive phosphate on an aspartate residue from histidine protein kinases known as sensor kinases (1,20). The histidine protein kinases autophosphorylate on a histidine residue and then serve as a phosphate donor to a cognate response regulator. Unlike most response regulators, Spo0A does not receive phosphate directly from a histidine protein kinase. Rather, phosphorylation of Spo0A requires the transfer of phosphate through the phosphorelay (5). The sporulation kinases first serve as phosphate donors to the response regulator encoded by spo0F. Phosphate is then transferred from Spo0F to Spo0B and finally to Spo0A (5, 7). The physiological function of the phosphorelay seems to be to integrate the many signals that regulate the initiation of sporulation (8, 10-13) and to generate a threshold concentration of Spo0AϳP necessary for the initiation of sporulation (6).Spo0AϳP is a transcriptional activator and repressor, depending on the location of the target binding site. Spo0AϳP represses transcription of abrB (22, 27) and activates transcription of several sporulation genes, including spoIIA (28), spoIIE (32), and spoIIG (3, 4). The abrB gene product is a transcriptional repressor, and repression of abrB by Spo0AϳP causes activation of genes that are normally repressed by AbrB (31,34). Less Spo0AϳP is needed for repression of abrB than for activation of the spoII genes, since several mutations that decrease activation of Spo0A have little or no effect on abrB expression while causing a decrease in expression of spoII genes (6,19,21,26,29).Three histidine protein kinases are involved in the initiation of sporulation: KinA (2, 21), KinB (29), and KinC (15,16). Under most sporulation conditions tested, kinA null mutations cause a small reduction in the sporulation frequency while null mutations in either kinB or kinC cause little or no decrease in sporulation frequency (15,16,29). The experiments described

The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.