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The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.

1. We have done cross-hybridizations between the mitochondrial ribosomal RNAs and DNAs from strains ST and PP of Tetrahymena pyriformis. DNA . ribosomal RNA hybrid formation can be completely prevented by an excess of the heterologous ribosomal RNA and the heterologous hybrids melt 6 degrees C below the homologous hybrids. This shows that the ribosomal RNA cistrons can account for the 5% cross-hybridization previously observed between the mtDNAs of strains PP and ST (Goldbach et al. (1977) Biochim. Biophys. Acta 477, 37--50). 2. By electron microscopy of DNA . ribosomal RNA hybrids we have determined the position of the ribosomal RNA cistrons on the mtDNA of strain GL, a mtDNA which we have shown to contain a sub-terminal 1 micron duplication-inversion and a terminal palindrome at one end which varies in length from 0 to 5 micron and which includes the 1 micron duplication-inversion (Arnberg et al. (1977) Biochim. Biophys. Acta 477, 51--69). The 21 S ribosomal RNA cistron overlaps the 1 micron duplication-inversion and as a result two or three cistrons are present, depending on the size of the terminal palindrome. Only one 14 S ribosomal RNA cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron and with the same polarity as this cistron. 3. We conclude from these results and those in the preceding paper that the sequence of the ribosomal RNAs and the position of the ribosomal RNA genes in the mtDNA is strongly conserved in Tetrahymena. Possible reasons for the duplication of 21-S ribosomal RNA genes and the terminal heterogeneity of Tetrahymena mtDNA are discussed.

1. Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex 'eye' of variable size in the middle. This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B. and Borst, P. (1974) Biochim. Biophys. Acta 361, 266-276). The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J. and Borst, P. (1978) Biochim. Biophys. Acta 521, 187-197). 2. Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends. mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%). The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 micron, most loops measuring 0.25--0.40 micron. No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends. 3. When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5'-termini with T4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, greater than 70% of the 3H and less than 30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands. Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15 degrees C did not lead to preferential labelling of terminal EcoRI fragments of the DNA. From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3'-OH groups. 4. A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication. This model can also account for the pronounced terminal heterogeneity previously observed in this DNA.