Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Binding Site Recognition and Docking Dynamics of a Single Electron Transport Protein: Cytochrome c2.

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to their redox partners and integral membrane proteins and exchanging electrons. Diffusive search, recognition, binding, and unbinding of these proteins often amount to kinetic bottlenecks in cellular energy conversion, but despite the availability of structures and intense study, the physical mechanisms controlling redox partner interactions remain largely unknown. The present molecular dynamics study provides an all-atom description of the cytochrome c2-docked bc1 complex in Rhodobacter sphaeroides in terms of an ensemble of favorable docking conformations and reveals an intricate series of conformational changes that allow cytochrome c2 to recognize the bc1 complex and bind or unbind in a redox state-dependent manner. In particular, the role of electron transfer in triggering a molecular switch and in altering water-mediated interface mobility, thereby strengthening and weakening complex formation, is described. The results resolve long-standing discrepancies between structural and functional data.

Molecular organization of cytochrome c2 near the binding domain of cytochrome bc1 studied by electron spin-lattice relaxation enhancement.

Measurements of specific interactions between proteins are challenging. In redox systems, interactions involve surfaces near the attachment sites of cofactors engaged in interprotein electron transfer (ET). Here we analyzed binding of cytochrome c2 to cytochrome bc1 by measuring paramagnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c2. PRE was exclusively induced by the iron atom of heme c1 of cytochrome bc1, which guaranteed that only the configurations with SL to heme c1 distances up to ∼30 Šwere detected. Changes in PRE were used to qualitatively and quantitatively characterize the binding. Our data suggest that at low ionic strength and under an excess of cytochrome c2 over cytochrome bc1, several cytochrome c2 molecules gather near the binding domain forming a "cloud" of molecules. When the cytochrome bc1 concentration increases, the cloud disperses to populate additional available binding domains. An increase in ionic strength weakens the attractive forces and the average distance between cytochrome c2 and cytochrome bc1 increases. The spatial arrangement of the protein complex at various ionic strengths is different. Above 150 mM NaCl the lifetime of the complexes becomes so short that they are undetectable. All together the results indicate that cytochrome c2 molecules, over the range of salt concentration encompassing physiological ionic strength, do not form stable, long-lived complexes but rather constantly collide with the surface of cytochrome bc1 and ET takes place coincidentally with one of these collisions.

Nano-mechanical mapping of the interactions between surface-bound RC-LH1-PufX core complexes and cytochrome c 2 attached to an AFM probe.

Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c 2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c 2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c 2 is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c 2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.

Nonredundant roles for cytochrome c2 and two high-potential iron-sulfur proteins in the photoferrotroph Rhodopseudomonas palustris TIE-1.

The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c2. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c2 resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c2. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c2 is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.

A critical role for the cccA gene product, cytochrome c2, in diverting electrons from aerobic respiration to denitrification in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.

Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

Tyrosine triad at the interface between the Rieske iron-sulfur protein, cytochrome c1 and cytochrome c2 in the bc1 complex of Rhodobacter capsulatus.

A triad of tyrosine residues (Y152-154) in the cytochrome c(1) subunit (C1) of the Rhodobacter capsulatus cytochrome bc(1) complex (BC1) is ideally positioned to interact with cytochrome c(2) (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations-A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation.

CcmI subunit of CcmFHI heme ligation complex functions as an apocytochrome c chaperone during c-type cytochrome maturation.

Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c(2) as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c(2) but not holocytochrome c(2). Mainly the C-terminal portions of both CcmI and apocytochrome c(2) mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c(2), like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c(2), but this interaction requires a free thiol group at apocytochrome c(2) heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae.

Cytochrome c-554 from Methylosinus trichosporium OB3b; a protein that belongs to the cytochrome c2 family and exhibits a HALS-Type EPR signal.

A small soluble cytochrome c-554 purified from Methylosinus trichosporium OB3b has been purified and analyzed by amino acid sequencing, mass spectrometry, visible, CD and EPR spectroscopies. It is found to be a mono heme protein with a characteristic cytochrome c fold, thus fitting into the class of cytochrome c(2), which is the bacterial homologue of mitochondrial cytochrome c. The heme iron has a Histidine/Methionine axial ligation and exhibits a highly anisotropic/axial low spin (HALS) EPR signal, with a g(max) at 3.40, and ligand field parameters V/ξ = 0.99, Δ/ξ = 4.57. This gives the rhombicity V/Δ = 0.22. The structural basis for this HALS EPR signal in Histidine/Methionine ligated hemes is not resolved. The ligand field parameters observed for cytochrome c-554 fits the observed pattern for other cytochromes with similar ligation and EPR behaviour.

Reorientation of cytochrome c2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer.

The reaction of water-soluble cytochrome c (c(2)) with its physiological redox partners is facilitated by electrostatic attractions between the two protein surfaces. Using spin-labeled cytochrome c(2) from Rhodobacter capsulatus and pulse electron paramagnetic resonance (EPR) measurements we compared spatial orientation of cytochrome c(2) upon its binding to surfaces of opposite charge. We observed that cytochrome c(2) can use its negatively charged "back" side when exposed to interact with positively charged surfaces (DEAE resin) which is the opposite to the use of its positively charged "front" side in physiological interaction with negatively charged binding domain of cytochrome bc(1). The later orientation is also adopted upon non-physiological binding of cytochrome c(2) to negatively charged carboxymethyl cellulose resin. These results directly demonstrate how the electric dipolar nature of cytochrome c(2) influences its orientation in interactions with charged surfaces, which may facilitate collisions with other redox proteins in a proper orientation to support physiologically-competent electron transfer. Saturation recovery EPR provides an attractive tool for monitoring spatial orientation of proteins in their interaction with surfaces in liquid phase. It is particularly valuable for metalloproteins engaged in redox reactions as a means to monitor the geometry and dynamics of formation of protein complexes in measurements that are independent of electron transfer processes.

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Organization of the electron transfer chain to oxygen in the obligate human pathogen Neisseria gonorrhoeae: roles for cytochromes c4 and c5, but not cytochrome c2, in oxygen reduction.

Although Neisseria gonorrhoeae is a prolific source of eight c-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes c(2), c(4), and c(5), encoded by the genes cccA, cycA, and cycB, respectively, have been investigated. Single mutations in genes for either cytochrome c(4) or c(5) resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible copy of the cycA or cycB gene. In contrast, a cccA mutant reduced oxygen slightly more rapidly than the parent, suggesting that cccA is expressed but cytochrome c(2) is not involved in electron transfer to cytochrome oxidase. The deletion of cccA increased the sensitivity of the cycB mutant to excess oxygen but decreased the sensitivity of the cycA mutant. Despite many attempts, a double mutant defective in both cytochromes c(4) and c(5) could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible cycB gene with deletions in both cycA and cycB was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome c(4) or c(5). These results define the gonococcal electron transfer chain to oxygen in which cytochromes c(4) and c(5), but not cytochrome c(2), provide alternative pathways for electron transfer from the cytochrome bc(1) complex to the terminal oxidase cytochrome cbb(3).

Identification, functional studies, and genomic comparisons of new members of the NnrR regulon in Rhodobacter sphaeroides.

Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain. nnrV, under control of a previously identified NnrR site, was also identified. paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase. paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome c(2) together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions.

Electron transfer from cytochrome c(2) to the reaction center: a transition state model for ionic strength effects due to neutral mutations.

Interprotein electron transfer plays an important role in biological energy conversion. In this work, the electron transfer reaction between cytochrome c(2) (cyt) and the reaction center (RC) was studied to determine the mechanisms coupling association and electron transfer. Previous studies have shown that mutation of hydrophobic residues in the reaction interface, particularly Tyr L162, changes the binding affinity and rates of electron transfer at low ionic strengths. In this study, the effect of ionic strength on the second-order electron transfer rate constant, k(2), between cyt c(2) and native or mutant RCs was examined. Mutations of hydrophobic and hydrogen bonding residues caused k(2) to decrease more rapidly with an increase in ionic strength. This change is explained with a transition state model by a switch from a diffusion-limited reaction in native RCs, where electron transfer occurs upon each binding event, to a fast exchange reaction in the Tyr L162 mutant, where dissociation occurs before electron transfer and k(2) depends upon the equilibrium between bound and free protein complexes. The difference in ionic strength dependence is attributed to a smaller effect of ionic strength on the energy of the transition state compared to the bound state due to larger distances between charged residues in the transition state. This model explains the faster dissociation rate at higher ionic strengths that may assist rapid turnover that is important for biological function. These results provide a quantitative model for coupling protein association with electron transfer and elucidate the role of short-range interactions in determining the rate of electron transfer.

Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells.

A procedure has been developed for directly depositing membrane fragments derived from bacterial cells (chromatophores from Rhodopseudomonas sphaeroides) and mammalian cells (mu-opioid receptor- and MC4 receptor-transfected human embryonic kidney (HEK) cells and rat trigeminal ganglion cells) on the silica surface of a plasmon-waveguide resonance (PWR) spectrometer. Binding of ligands (cytochrome c(2) for the chromatophores, the peptide agonists DAMGO and melanotan-II that are specific for the mu-opioid and MC4 receptors, and two nonpeptide agonists that are specific for the CB1 receptor) to these membrane fragments has been observed and characterized with high sensitivity using PWR spectral shifts. The K(D) values obtained are in excellent agreement with conventional pharmacological assays and with prior PWR studies using purified receptors inserted into deposited lipid bilayer membranes. These studies provide a new tool for obtaining useful biological information about receptor-mediated processes in real biological membranes.

Interaction between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: role of interprotein hydrogen bonds in binding and electron transfer.

The role of short-range hydrogen bond interactions at the interface between electron transfer proteins cytochrome c(2) (cyt) and the reaction center (RC) from Rhodobacter sphaeroides was studied by mutation (to Ala) of RC residues Asn M187, Asn M188, and Gln L258 which form interprotein hydrogen bonds to cyt in the cyt-RC complex. The largest decrease in binding constant K(A) (8-fold) for a single mutation was observed for Asn M187, which forms an intraprotein hydrogen bond to the key residue Tyr L162 in the center of the contact region with a low solvent accessibility. Interaction between Asn M187 and Tyr L162 was also implicated in binding by double mutation of the two residues. The hydrogen bond mutations did not significantly change the second-order rate constant, k(2), indicating the mutations did not change the association rate for formation of the cyt-RC complex but increased the dissociation rate. The first-order electron transfer rate, k(e), for the cyt-RC complex was reduced by a factor of up to 4 (for Asn M187). The changes in k(e) were correlated with the changes in binding affinity but were not accompanied by increases in activation energy. We conclude that short-range hydrogen bond interactions contribute to the close packing of residues in the central contact region between the cyt and RC near Asn M187 and Tyr L162. The close packing contributes to fast electron transfer by increasing the rate of electronic coupling and contributes to the binding energy holding the cyt in position for times sufficient for electron transfer to occur.

A mechanistic and electrochemical study of the interaction between dimethyl sulfide dehydrogenase and its electron transfer partner cytochrome c2.

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron-sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c2 mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c2 accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c2), Michaelis constants, KM,DMS and KM,cyt of 53 and 21 microM, respectively, were determined at pH 8. Further kinetic analysis revealed a "ping-pong" enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c2 immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible FeIII/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The FeIII/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c2 is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c2 effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant K'M,DMS of 13(+/-1) microM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c2 as the electron acceptor was found to be approximately 8.3.

Conformational control of the Q(A) to Q(B) electron transfer in bacterial reaction centers: evidence for a frozen conformational landscape below -25 degrees C.

The competition between the P(+)Q(A)(-) --> PQ(A) charge recombination (P, bacteriochlorophyll pair acting as primary photochemical electron donor) and the electron transfer to the secondary quinone acceptor Q(A)(-)Q(B) --> Q(A)Q(B)(-) (Q(A) and Q(B), primary and secondary electron accepting quinones) was investigated in chromatophores of Rb. capsulatus, varying the temperature down to -65 degrees C. The analysis of the flash-induced pattern for the formation of P(+)Q(A)Q(B)(-) shows that the diminished yield, when lowering the temperature, is not due to a homogeneous slowing of the rate constant k(AB) of the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer but to a distribution of conformations that modulate the electron transfer rate over more than 3 orders of magnitude. This distribution appears "frozen", as no dynamic redistribution was observed over time ranges > 10 s (below -25 degrees C). The kinetic pattern was analyzed to estimate the shape of the distribution of k(AB), showing a bell-shaped band on the high rate side and a fraction of "blocked" reaction centers (RCs) with very slow k(AB). When the temperature is lowered, the high rate band moves to slower rate regions and the fraction of blocked RCs increases at the expense of the high rate band. The RCs that recombine from the P(+)Q(A)Q(B)(-) state appear temporarily converted to a state with rapid k(AB), indicating that the stabilized state described by Kleinfeld et al. (Biochemistry 1984, 23, 5780-5786) is still accessible at -60 degrees C.

Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling.

Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (NO(2)(-))-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the NO(2)(-)-dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on NO(2)(-)-dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells.