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The effects of protein dehydration upon the equilibrium and dynamic properties of the heme active site in human hemoglobin (HbA) have been probed by resonance Raman scattering. Spectra of equilibrium carbonmonoxy-HbA and the photolytic heme transient species generated within 10 ns of ligand photolysis have been obtained from thin films of protein in various stages of dehydration. These data provide detailed information concerning the response of the heme and its bonding interactions with both the proximal histidine and carbon monoxide as a function of protein hydration. For protein hydration levels of 0.4-1.0 g of H2O/g of protein, our results indicate that the C = O stretching mode of carbonmonoxy-HbA is dramatically affected by protein hydration levels, thus corroborating the infrared results of Brown et al. [Brown, W. E., Sutcliffe, J. W., & Pulsinelli, P. D. (1983) Biochemistry 22, 2914-2923]. However, we find that both heme skeletal modes and the Fe-C bond strength are largely insensitive to dehydration. Moreover, the proximal pocket geometry (as reflected in the behavior of the Fe-proximal histidine stretching mode) immediately following ligand photolysis was found to be very similar to that of R-state solution hemoglobin. At protein hydration levels below the theoretical monolayer limit, small changes in the resonance Raman spectra of both equilibrium HbCO and the transient heme species generated subsequent to ligand photolysis are detected. These include broadening of the Fe-C stretching mode in equilibrium HbCO and a small shift to lower frequency of the Fe-His mode in the photolytic transient species.(ABSTRACT TRUNCATED AT 250 WORDS)

The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)

The three major monomer hemoglobins from Glycera dibranchiata erythrocytes isolated in this laboratory were sequenced from their N-termini. A stretch of amino acid sequence identity was used to determine the sequence of a mixed oligodeoxynucleotide that would be complementary to all 12 possible mRNA sequences coding for the amino acids. A cDNA library was constructed by using poly(A+) RNA from G. dibranchiata erythrocytes, the library was probed with the oligonucleotide, and the longest positive inserts found were subcloned into a sequencing plasmid and then sequenced. The first one was 745 bases long, containing 85 bases of 5'-untranslated RNA, an open reading frame of 444 bases coding for 148 amino acids, and a 3'-untranslated region of 216 bases. The predicted amino acid sequence matches the first 25 amino acids of G. dibranchiata monomer globin component IV. The sequence contains an N-terminal methionine plus 18 other mostly conservative sequence changes compared to the published sequence of Imamura et al. (1972), which appears from our partial sequencing to be monomer globin component II. We confirm the presence of leucine in the E7 position, which is histidine in most myoglobins and hemoglobins.