The Alkaline Transition of Bis(N-acetylated) Heme Undecapeptide.

Alkaline forms of the ferric bis(N-acetylated) heme undecapeptide of cytochrome c (N-ac-HUP) and some of its derivatives have been studied by electronic absorption and electron paramagnetic resonance spectroscopies. Surprisingly, even at pH >12, no evidence could be found for the formation of a hydroxyl ion adduct, in direct contrast to a previous report concerning ferric heme peptides encapsulated in detergent micelles (Mazumdar et al. Inorg. Chem. 1991, 30, 700-705). A spectroscopically determined pK(a) of approximately 9 is assigned to the deprotonation of the constituent histidine ligand of heme iron in N-ac-HUP. The present findings are not entirely in keeping with those of an earlier study concerning the properties of N-acetylated heme octapeptide (Wang et al. J. Biol. Chem. 1992, 35, 15310-15318), the differences observed being attributed to the buffering media employed in the two investigations. The implications of the current results in relation to a better understanding of the alkaline transitions observed in hemoglobins and myoglobins is considered.

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.

In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wild-type. However, less efficient packing of residues in the mutant surrounding the heme propionates leads to more solvent exposure for both propionate groups, which may account for decreased stability of the mutant. The mutant has a reduction potential different from wild-type, and furthermore, the pH dependence of this potential is not the same as for wild-type. The structure of the mutant suggests that these changes are related to the loss of the residue-47 propionate hydrogen bond and the loss of charge on the side chain of residue 47.

Expression of Pseudomonas stutzeri Zobell cytochrome c-551 and its H47A variant in Escherichia coli.

The nirM gene encoding cytochrome c-551 from Pseudomonas stutzeri Zobell (PZ) has been expressed in Escherichia coli at levels higher than those previously reported but only under strict anaerobic growth conditions. Expression yields for wild-type cytochrome in this study typically reached 0.6 micromol per liter of saturated E. coli culture (5.5mg/L). Culture conditions investigated are compared to obtained c-551 expression levels; the results may lead to a greater understanding of the challenges encountered when expressing c-type hemoproteins in E. coli. The nirM gene was mutated to produce a histidine-47-alanine mutation of c-551 that been heterologously expressed in E. coli using optimum culture conditions and had its physiochemical properties compared to those of the wild-type protein. In PZ, the histidine-47 residue is part of a conserved hydrogen-bonding network located at the bottom of the heme crevice that also involves tryptophan-56 and a heme propionate. Ionization events within this network are experimentally demonstrated to modulate c-551 oxidation-reduction potential and its observed dependence on pH around neutrality. The redox potential of the mutant cytochrome still displays pH-dependence; however, the midpoint potential is approximately 25mV lower with respect to wild-type c-551 at neutral pH while the pK at which the heme propionate (HP-17) ionizes is lowered by 1.3 pH units. Temperature and chemical denaturant studies also show that loss of the hydrogen-bond-donating imidazole leads to a large decrease in c-551 tertiary stability.