The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.

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.

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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.

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The gene encoding for bacterial cytochrome c-551 from Pseudomonas stutzeri substrain ZoBell has been mutated to convert the invariant sixth ligand methionine residue into histidine, creating the site-specific mutant M61H. Proton NMR resonance assignments were made for all main-chain and most-side chain protons in the diamagnetic, reduced form at pH 9.2 and 333 K by two-dimensional NMR techniques. Distance constraints (1074) were determined from nuclear Overhauser enhancements and main-chain torsion-angle constraints (72) from scalar coupling estimates. Solution conformations for the protein were computed by the simulated annealing approach. For 28 computed structures, the root mean squared displacement from the average structure excluding the terminal residues 1, 2, 81, and 82 was 0.52 A (sigma = 0.096) for backbone atoms and 0.90 A (sigma = 0.122) for all heavy atoms. The global folding of the mutant protein is the same as for wild type. The biggest changes are localized in a peptide span over residues 60-65. The most striking behavior of the mutant protein is that at room temperature and neutral pH it exists in a state similar to the molten globular state that has been described for several proteins under mild denaturing conditions, but the mutant converts to a more ordered state at high pH and temperature.