The binding interface of cytochrome c and cytochrome c₁ in the bc₁ complex: rationalizing the role of key residues

The interaction of cytochrome c with ubiquinol-cytochrome c oxidoreductase (bc₁ complex) has been studied for >30 years, yet many aspects remain unclear or controversial. We report the first molecular dynamic simulations of the cyt c-bc₁ complex interaction. Contrary to the results of crystallographic studies, our results show that there are multiple dynamic hydrogen bonds and salt bridges in the cyt c-c₁ interface. These include most of the basic cyt c residues previously implicated in chemical modification studies. We suggest that the static nature of x-ray structures can obscure the quantitative significance of electrostatic interactions between highly mobile residues. This provides a clear resolution of the discrepancy between the structural data and functional studies. It also suggests a general need to consider dynamic interactions of charged residues in protein-protein interfaces. In addition, a novel structural change in cyt c is reported, involving residues 21-25, which may be responsible for cyt c destabilization upon binding. We also propose a mechanism of interaction between cyt c₁ monomers responsible for limiting the binding of cyt c to only one molecule per bc₁ dimer by altering the affinity of the cytochrome c binding site on the second cyt c₁ monomer.     

The complex of cytochrome c and cytochrome c peroxidase: the end of the road?

Cytochrome c (Cc) and cytochrome c peroxidase (CcP) form a physiological complex in the inter-membrane space of yeast mitochondria, where CcP reduces hydrogen peroxide to water using the electrons provided by ferrous Cc. The Cc-CcP system has been a popular choice of study of interprotein biological electron transfer (ET) and in understanding dynamics within a protein-protein complex. In this review we have charted seven decades of research beginning with the discovery of CcP and leading to the latest functional and structural work, which has clarified the mechanism of the intermolecular ET, addressed the putative functional role of a low-affinity binding site, and identified lowly-populated intermediates on the energy landscape of complex formation. Despite the remarkable attention bestowed on this complex, a number of outstanding issues remain to be settled on the way to a complete understanding of Cc-CcP interaction.     

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 c₁ subunit (C1) of the Rhodobacter capsulatus cytochrome bc₁ complex (BC1) is ideally positioned to interact with cytochrome c₂ (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.     

Intermonomer electron transfer between the low-potential b hemes of cytochrome bc₁

Cytochrome (cyt) bc(1) is a structural dimer with its monomers consisting of the Fe-S protein, cyt b, and cyt c(1) subunits. Its three-dimensional architecture depicts it as a symmetrical homodimer, but the mobility of the head domain of the Fe-S protein indicates that the functional enzyme exists in asymmetrical heterodimeric conformations. Here, we report a new genetic system for studying intra- and intermonomer interactions within the cyt bc(1) using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc(1) structural genes carried by two plasmids that are coharbored by a cell without its endogenous enzyme. Our results indicate that coexpressed cyt bc(1) subunits were matured, assorted, and assembled in vivo into homo- and heterodimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of intermonomer electron transfer between the low-potential b hemes of cyt bc(1) was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites of the enzyme. The data demonstrate that active heterodimeric variants, formed of monomers carrying mutations that abolish only one of the two (Q(o) or Q(i)) active sites of each monomer, are produced, and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and heterodimeric cyt bc(1) preparations, demonstrated that efficient and productive electron transfer occurs between the low-potential b(L) hemes of the monomers in a heterodimeric enzyme. Overall findings are discussed with respect to intra- and intermonomer interactions that take place during the catalytic turnover of cyt bc(1).     

Co-evolution of cytochrome c 6 and plastocyanin, mobile proteins transferring electrons from cytochrome b 6f to photosystem I

 Cytochrome c ₆ and plastocyanin are soluble metalloproteins that act as mobile carriers transferring electrons between the two membrane-embedded photosynthetic complexes cytochrome b ₆ f and photosystem I (PSI). First, an account of recent data on structural and functional features of these two membrane complexes is presented. Afterwards, attention is focused on the mobile heme and copper proteins – and, in particular, on the structural factors that allow recognition and confer molecular specificity and control the rates of electron transfer from and to the membrane complexes. The interesting question of why plastocyanin has been chosen over the ancient heme protein is discussed to place emphasis on the evolutionary aspects. In fact, cytochrome c ₆ and plastocyanin are presented herein as an excellent case study of biological evolution, which is not only convergent (two different structures but the same physiological function), but also parallel (two proteins adapting themselves to vary accordingly to each other within the same organism). Received: 4 July 1996 / Accepted: 16 September 1996     

Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

Physiology,Hygiene and the Entry of Women to the Medical Profession in Edinburgh c. 1869–c. 1900

Academic physiology, as it was taught by John Hughes Bennett during the 1870s, involved an understanding of the functions of the human body and the physical laws which governed those functions. This knowledge was perceived to be directly relevant and applicable to clinical practice in terms of maintaining bodily hygiene and human health. The first generation of medical women received their physiological education at Edinburgh University under Bennett, who emphasised the importance of physiology for women due to its relevance for the hygienic needs of the family and of society. With the development of laboratory-based science as a distinct aspect of medical education during the later nineteenth century, however, so the direct application of physiology to clinical practice diminished. The understanding of physiology as hygiene was marginalised by the new orthodoxy of scientific medicine. This shift in the physiological paradigm enabled medical women to stake out a specific field of interest within medicine which was omitted from the new definition of physiology as pure medical science: hygiene and preventive medicine. Women physicians were able to take advantage of the shift towards science as the basis of medical theory and practice to define their own specific role within the profession.     

Differences in the dynamics of oxidized and reduced cytochrome c measured by Mössbauer spectroscopy

The temperature dependence of the mean square displacement of the iron atom in reduced and oxidized cytochrome c has been studied by Mössbauer spectroscopy. The flexibility of the protein, labeled by the modes coupling to the iron, is diminished upon reduction. The differences in flexibility are sufficient to explain the differences in physicochemical properties between the oxidized and the reduced forms.     

The pH dependence of the stereochemistry around the heme group in NO–cytochrome c (horse heart)

The electronic absorption, EPR and MCD spectra of NO derivatives of both ferrous and ferric cytochrome c (horse heart) have been measured in the pH region 2.0 to 12.9, in order to elucidate the pH dependence of the stereochemistry around the heme group. The reaction products of NO with ferrous cytochrome c in equilibrium were as follows: in the region 2.0 ⩽ pH ⩽ 5.3, NO–ferrous cytochrome c; in the region 5.3 < pH ⩽ 11.0, a mixture of NO–ferrous cytochrome c and native ferrous cytochrome c; at pH 12.0, NO–ferrous cytochrome c. At pH 2.0, the NO–ferrous cytochrome c contained a five-coordinate nitrosylheme as the major component and a six-coordinate species as the minor component, and at the order pH values it contained only the six-coordinate species. The reaction products of NO with ferric cytochrome c in equilibrium were as follows: in the region 2.0 ⩽ pH ⩽ 7.2, NO–ferric cytochrome c with six-coordinate nitrosylheme; in the region 7.2 < pH ⩽ 11.0, a mixture of NO–ferrous cytochrome c and native ferrous cytochrome c; at pH 12.0, NO–ferrous cytochrome c. Thus, the reaction of NO with ferric cytochrome c results in the formation of NO–ferrous cytochrome c, which is a typical case of reductive nitrosylation.     

Are there isoenzymes of cytochrome c oxidase in Paracoccus denitrificans?

We have used a gene replacement strategy to delete the previously isolated gene [(1987) EMBO J. 6, 2825-2833] for the cytochrome c oxidase subunit I from Paracoccus denitrificans. The resulting mutant was still able to synthesize active cytochrome c oxidase. This led us to look for another locus which could completely suppress the mutation. In this study we report the isolation of a second gene encoding subunit I. An open reading frame coding for cytochrome c 550 was found upstream from this gene. We suggest that there are isoenzymes of cytochrome c oxidase (cytochrome aa3) in this bacterium.     

Do evolutionary changes in cytochrome c structure reflect functional adaptations?

Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

Stability diagram and unfolding of a modified cytochrome c: what happens in the transformation regime?

We determined the stability diagram of a modified cytochrome c protein in a glycerol water mixture by measuring the first and the second moment of the fluorescence from the chromophore as a function of temperature and pressure. Temperature and pressure were varied between 273 and 363 K and 0.0001 and 1 GPa, respectively. The shift of the fluorescence maximum showed a characteristic sigmoid-like pattern from which information on the microscopic processes during unfolding is obtained: as the transformation regime is entered, the fluorescence shows a significant blue shift. The conclusion is that water molecules get into contact with the chromophore. They lead to strong electrostatic contributions in the solvent shift, which counteract the red shifting dispersion interactions. Assuming that there are just two relevant states that determine the stability diagram, the complete set of thermodynamic parameters can be determined from the data. However, under certain pressure-temperature conditions the fluorescence pattern is more complicated, pointing toward reentrant transitions and, possibly, to consecutive steps in the unfolding process.     

Domain conformational switch of the iron–sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc₁ complex in the past have led to the formulation of the “protonmotive Q-cycle” mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q_P site with both electrons transferred simultaneously to ISP and cyt b_L when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc₁ demonstrates that the reduced ISP-ED moves to the c₁-position to reduce cyt c₁ only after the reduced cyt b_L is oxidized by cyt b_H. However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q_P site inhibitors, Pm and Pf, under various redox states of the bc₁ complex, suggest that the electron transfer from heme b_L to b_H is the driving force for the releasing of the reduced ISP-ED from the b-position to c₁-position to reduce cyt c₁.     

Microsomal monooxygenase in apoptosis: another target for cytochrome c signaling?

The main function of eukaryotic microsomal monooxygenase (MMO) is thought to be the oxygenation of xenobiotics and hydrophobic endogenous substrates. However, there are important inconsistencies in the concept that the biological role of MMO is limited to the catalysis of these reactions. It is probable that MMO is also a regulated generator of reactive oxygen species (ROS) that is involved in both the initiation and execution of apoptosis. Additional support for this hypothesis came with the discovery of a role for cytochrome c (cyt c) in apoptotic signaling. This article introduces the theory that microsomal cytochrome b(5), which modulates the production of ROS in MMO, is among the principal interacting targets of cyt c. The role of this interaction in the initiation of apoptosis is discussed.     

Coupled electron and proton transfer reactions during the O→E transition in bovine cytochrome c oxidase

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O→E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK(a)s and E(m)s of the key ionizable and redox-active groups at the different stages of the O→E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O→E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO.     

Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 : primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c 3

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8?Å electron density maps. The refinement at 1.8?Å resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c ₃, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229–514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c ₃ from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c ₃ and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c ₃ upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.     

Refuge from predation, the benefit of living in an extreme acidic environment?

Organisms living in extreme habitats require costly adaptations to cope with these conditions. Among the suggested potential benefits that trade off these costs is refuge from predation. To study these interactions in extreme environments, samples were taken in the cave Cueva de Villa Luz, Tabasco, Mexico, where more than 32 subterranean springs, some H(2)S rich, rise from the floor. Hydrogen sulfide gas plus oxygen is absorbed by freshwater, and oxidation forms concentrated sulfuric acid. Snottites, whitish hollow mucous tubes, hang from the ceiling of the cave. Fluid drops from these snottites were recorded as having pH values of 0-3. We report the discovery of a new species of nematode that thrives in the highly acidic environment of the snottite. Micro CT scan of snottites reveals a complex interaction between the acidic snottite, nematodes, and abundant nematode-eating mites. The nematode adaptation to low pH probably protects them against mite predation, for which nematodes are most likely the most important source of carbon in this sulfur-driven ecosystem.