The alkaline conformation (state IV) of yeast iso-1-ferricytochrome c and variants in which selected lysyl residues were replaced with alanyl residues has been studied by 1H NMR spectroscopy, electronic spectroscopy, EPR spectroscopy, direct electrochemistry, pH-jump kinetics, and temperature-dependent circular dichroism spectroscopy. On the basis of the NMR studies, Lys73 and Lys79 are shown to replace Met80 as the axial ligand in the two conformers of state IV that were detected in previous studies (Hong, X. L.; Dixon, D. W. FEBS Lett. 1989, 246, 105−108; Ferrer, J. C.; Guillemette, J. G.; Bogumil, R.; Inglis, S. C.; Smith, M.; Mauk, A. G. J. Am. Chem. Soc. 1993, 115, 7507−7508). The pK a for the conformational equilibrium between state III (native conformation) and state IV of the wild-type protein (8.70(2)) is found to be intermediate between that of the Lys73 bound conformer (8.44(1)) and that of the Lys79 bound conformer (8.82(2)) (0.1 M NaCl, 25 °C) as are the kinetic parameters for the conversion of native protein to each of the two alkaline conformers and the midpoint reduction potentials of the two alkaline forms. The EPR spectra of the Lys73Ala and Lys79Ala variants permit interpretation of the corresponding spectrum of the wild-type protein as the sum of two component conformers. The Lys79Ala variant is slightly more susceptible to thermal denaturation at pH 6.15, but the Lys73Ala variant is less thermally stable than the wild-type cytochrome or the Lys79Ala variant at alkaline pH. The Lys73Ala/Lys79Ala double variant retains the spectroscopic characteristics of the native cytochrome at moderately high pH and appears to undergo a change of axial ligation only under more alkaline conditions (pK a ∼ 10.5). This observation suggests that the coordination of the amine ligands is a significant contribution toward the driving force for formation of the state IV conformers. These results establish the axial ligation of yeast iso-1-ferricytochrome c state IV, characterize the kinetics with which state III converts to state IV, and establish the electrochemical properties and thermal stabilities of the two conformers that constitute state IV. The results of this work are discussed with reference to pH-dependent structural behavior of other proteins, the mechanism by which these conformers of the ferricytochrome are formed, and the relationship of the present results to those reported previously for the formation of state IV from state III.

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant from the copper cofactor. Comparison of this structure with a refined nitrite-bound crystal structure explains how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The side-on copper-nitrosyl in nitrite reductase expands the possibilities for nitric oxide interactions in copper proteins such as superoxide dismutase and prions.

Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3). Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3. Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1. Both proteins were expressed in the cytoplasm of E. coli cells harboring the plasmid. Semianaerobic cultures grown in a fermentor produced 15 mg of recombinant iso-1-cytochrome c per liter of culture. Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population. Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E. coli was compared to the same protein expressed in yeast. At neutral pH, the two proteins exhibit indistinguishable spectroscopic and physical (Tm, Em') characteristics. However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E. coli as it is by S. cerevisiae. Interestingly, the pKa of the alkaline transition of the protein expressed in E. coli is approximately 0.6 pKa unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7). 1H NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast. These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine.

A specific DNA oligonucleotide--hemin complex (PS2.M--hemin complex) that exhibits DNA-enhanced peroxidative activity was studied by EPR and UV--visible spectroscopy and by chemical probing analysis. EPR data obtained from low-temperature experiments on the PS2.M--hemin complex showed both a low-field g approximately 6 and a high-field g approximately 2 signal. These EPR signals are typical of high-spin ferric heme with axial symmetry as judged by the EPR spectrum of six-coordinate heme iron in acidic Fe(III)-myoglobin. This similarity is consistent with the presence of two axial ligands to the heme iron within the PS2.M--hemin complex, one of which is a water molecule. Optical analyses of the acid-base transition for the hemin complex yielded a pK(a) value for the water ligand of 8.70 +/- 0.03 (mean +/- SD). Low-temperature EPR analysis coupled with parallel spin-trapping investigations following the reaction of the PS2.M--hemin complex and hydrogen peroxide (H(2)O(2)) indicated the formation of a carbon-centered radical, most likely on the PS2.M oligonucleotide. Chemical probing analysis identified specific guanine bases within the PS2.M sequence that underwent oxidative damage upon reaction with H(2)O(2). These and other experimental findings support the hypothesis that the interaction of specific guanines of PS2.M with the bound hemin cofactor might contribute to the superior peroxidative activity of the PS2.M--hemin complex.