Conformational toggling of yeast iso-1-cytochrome C in the oxidized and reduced states.

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined. The structures indicate that the half molecule of cyt c (aa 50-102) presents a kind of "zigzag riveting ruler" structure, residues at certain positions of this region such as Pro71, Lys73 can move a big distance by altering the tertiary structure while maintaining the secondary structures. This finding provides a molecular insight into conformational toggling in different oxidation states of cyt c that is principle significance to its biological functions in electron transfer and apoptosis. Structural analysis also reveals that Pro71 functions as a key hydrophobic patch in the folding of the polypeptide of the region (aa 50-102), to prevent heme pocket from the solvent.

Escherichia coli cytochrome c nitrite reductase NrfA.

The periplasmic cytochrome c nitrite reductase (Nrf) system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. Nitric oxide (NO) is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate. This chapter describes the method used to assay Nrf-catalyzed NO reduction in whole cells of E. coli and the procedures for preparing highly purified NrfA suitable for use in kinetic, spectroscopic, voltammetric, and crystallization studies.

Is cytochrome b glutamic acid 272 a quinol binding residue in the bc1 complex of Saccharomyces cerevisiae?

The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c coupled to a vectorial translocation of protons across the membrane. On the basis of the three-dimensional structures of the bc1 complex in the presence of the inhibitor stigmatellin, it was assumed that the substrate quinol binding involves the cyt b glutamate residue E272 and the histidine 181 on the Rieske protein. Although extensive mutagenesis of glutamate E272 has been carried out, different experimental results were recently obtained, and different conclusions were drawn to explain its role in the bifurcated electron/proton transfer at the QO site. This residue is not totally conserved during evolution. We show in this study that replacement of E272 with apolar residues proline and valine naturally present in some organisms did not abolish the bc1 activity, although it slowed down the kinetics of electron transfer. The Km value for the binding of the substrate quinol was not modified, and the EPR data showed that the quinone/quinol binding still occurred in the mutants. Binding of stigmatellin was retained; however, mutations E272P,V induced resistance toward the QO site inhibitor myxothiazol. The pH dependence of the bc1 activity was not modified in the absence of the glutamate E272. Our results suggest that this residue may not be involved in direct substrate binding or in its direct deprotonation. Revertants were selected from the respiratory deficient mutant E272P. The observed suppressor mutations introduced polar residues serine and threonine at position 272. The data lead us to suggest that E272 may be involved in a later step on the proton exit pathway via the interaction with a water molecule.

Physiological and gene expression analysis of inhibition of Desulfovibrio vulgaris hildenborough by nitrite.

A Desulfovibrio vulgaris Hildenborough mutant lacking the nrfA gene for the catalytic subunit of periplasmic cytochrome c nitrite reductase (NrfHA) was constructed. In mid-log phase, growth of the wild type in medium containing lactate and sulfate was inhibited by 10 mM nitrite, whereas 0.6 mM nitrite inhibited the nrfA mutant. Lower concentrations (0.04 mM) inhibited the growth of both mutant and wild-type cells on plates. Macroarray hybridization indicated that nitrite upregulates the nrfHA genes and downregulates genes for sulfate reduction enzymes catalyzing steps preceding the reduction of sulfite to sulfide by dissimilatory sulfite reductase (DsrAB), for two membrane-bound electron transport complexes (qmoABC and dsrMKJOP) and for ATP synthase (atp). DsrAB is known to bind and slowly reduce nitrite. The data support a model in which nitrite inhibits DsrAB (apparent dissociation constant K(m) for nitrite = 0.03 mM), and in which NrfHA (K(m) for nitrite = 1.4 mM) limits nitrite entry by reducing it to ammonia when nitrite concentrations are at millimolar levels. The gene expression data and consideration of relative gene locations suggest that QmoABC and DsrMKJOP donate electrons to adenosine phosphosulfate reductase and DsrAB, respectively. Downregulation of atp genes, as well as the recorded cell death following addition of inhibitory nitrite concentrations, suggests that the proton gradient collapses when electrons are diverted from cytoplasmic sulfate to periplasmic nitrite reduction.

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.

The iron-sulfur protein is necessary for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex of yeast mitochondria.

A strain of yeast lacking the gene for the Rieske iron-sulfur protein (RIP) of the cytochrome b-c1 complex was used to study the assembly of this complex in the mitochondrial membrane. This strain lacks the mRNA for the iron-sulfur protein as evidenced by both Northern hybridization using a probe containing the coding region of the gene plus in vitro translation of total RNA followed by immunoprecipitation with a specific antibody against the iron-sulfur protein. In addition, isolated mitochondria from this strain lacked cytochrome c reductase activity with either succinate or the decyl analog of ubiquinol as substrate. Immunoblotting studies with antiserum against the cytochrome b-c1 complex revealed that mitochondria from the iron-sulfur protein-deficient strain have levels of core protein I, core protein II, and cytochrome c1 equal to those of wild-type mitochondria; however, a decrease in cytochrome b was evident from both immunoblotting and spectral analysis. Moreover, it is evident from the immunoprecipitates of radiolabeled mitochondria that the amounts of the low-molecular-weight subunits (17, 14, and 11 kDa) are decreased 53, 65, and 50%, respectively, in mitochondria lacking the iron-sulfur protein. These results suggest that the iron-sulfur protein is required for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex.