Heme Cytochrome Research Articles

Structure of the bc1–cbb3 respiratory supercomplex from Pseudomonas aeruginosa

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc1) and one of the organism's terminal oxidases, cytochrome cbb3, which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c4 and a single heme cytochrome c5. Together, these proteins allow electron transfer from ubiquinol in cytochrome bc1 to oxygen in cytochrome cbb3. We also present evidence that different isoforms of cytochrome cbb3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc1-cbb3 SCs also exist in other bacterial pathogens.

Confocal Raman imaging reveals the impact of retinoids on human breast cancer via monitoring the redox status of cytochrome c

This paper expands the current state of knowledge on impact of retinoids on redox status of cytochrome c in cancers. Little is known how the expression of cytochromes may influence the development of cancers. We studied the effect of the redox status of the central iron ion in heme of cytochrome c. We determined the redox status of the iron ion in cytochrome c in mitochondria, cytoplasm, lipid droplets, and endoplasmic reticulum of the human breast cancer cells by Raman imaging. We incubated human breast adenocarcinoma cells (SK-BR-3) with retinoic acid, retinol and retinyl ester (palmitate) at concentration of 50 μM for 24 h. We recorded the Raman spectra and images of human breast cancer in vitro SK-BR-3 cells receiving redox stimuli by retinoic acid, retinol and retinyl ester (palmitate). The paper provides evidence that retinoic acid and retinol are pivotally important for mitochondrial energy homeostasis by controlling the redox status of cytochrome c in the electron transport chain controlling oxidative phosphorylation and apoptosis. We discussed the role of retinoids in metabolism and signaling of cancer cells. The paper provides experimental support for theoretical hypothesis how retinoic acid/retinol catalyse resonance energy transfer reactions and controls the activation/inactivation cycle of protein kinase PKCδ.

Hemoglobin and cytochrome c. reinterpreting the origins of oxygenation and oxidation in erythrocytes and in vivo cancer lung cells

Maintaining life (respiration), cell death (apoptosis), oxygen transport and immunity are main biological functions of heme containing proteins. These functions are controlled by the axial ligands and the redox status of the iron ion (oscillations between Fe2+ and Fe3+) in the heme group. This paper aims to evaluate the current state of knowledge on oxidation and oxygenation effects in heme proteins. We determined the redox status of the iron ion in whole blood (without and with anticoagulant), hemoglobin in erythrocytes, in isolated cytochrome c and cytochrome c in mitochondria of the human lung cancer cells using UV–VIS electronic absorption spectroscopy, Raman spectroscopy and Raman imaging. Here we discussed the mechanism responsible for the Q electronic absorption band spectral behavior, i.e., its splitting, and its change in extinction coefficient, as well as vibrational modifications upon oxygenation and oxidation. We compared the redox status of heme in hemoglobin of human erythrocytes and cytochrome c in mitochondria of human lung cancer cells. Presented results allow simultaneous identification of oxy- and deoxy-Hb, where 1547 and 1604 cm−1 vibrations correspond to deoxygenated hemoglobin, while 1585 and 1638 cm−1 correspond to oxyhemoglobin, respectively. Our results extend knowledge of oxidation and oxygenation effects in heme proteins. We demonstrated experimentally the mechanism of electronic-vibrational coupling for the Q band splitting. Presented results extend knowledge on oxidation and oxygenation effects in heme proteins and provide evidence that both processes are strongly coupled. We showed that retinoic acid affects the redox state of heme in cytochrome c in mitochondria. The change of the redox status of cytochrome c in mitochondria from the oxidized form to the reduced form has very serious consequences in dysfunction of mitochondria resulting in inhibition of respiration, apoptosis and cytokine induction.

SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential

The balance between the mitochondrial respiratory chain activity and the cell's needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for the lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We have verified that molecule bond vibrations assessed by SERS are a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have demonstrated that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential (ΔΨ) and H+ concentration in the intermembrane space. This regulates the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cell's consumption of ATP and the overall activity. We have found that under hypertensive conditions cytochrome c heme loses its sensitivity to ΔΨ that can affect the regulation of ETC activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens broad perspectives in cell bioenergetics.

Boosting bioelectricity generation in microbial fuel cells via biomimetic Fe-N-S-C nanozymes

Anode performance has been regarded as a crucial factor determining long-term stability and electricity generation of microbial fuel cells (MFCs), which restricts by the difficult extracellular electron transfer (EET) on the microbe/anode interface. Herein, inspired by biological enzyme systems, this study synthesized the biomimetic nanozymes with Fe-N-S-C active sites as the anode materials of MFCs, which was similar to the hemes of c-type cytochromes (c-Cyts) for boosting EET process. As excepted, an obviously faster start-up and a much higher power density were achieved by the MFCs equipped with Fe-N-S-C nanozymes (startup time, 3.5 d; power density, 2366 ± 34 mW m−2) than that based on traditional carbon cloth (startup time, 5.6 d; power density, 1009 ± 26 mW m−2). Such unique features of Fe-N-S-C nanozymes anode not only greatly favored the bacterial adhesion and the electroactive bacteria enrichment on the anode surface, but also efficiently facilitated the EET process between the electroactive bacteria and anode surface. This study provided a feasible strategy for designing the novel MFC anode materials from the perspective of bionic enzyme.

Cofactor Dynamics Couples the Protein Surface to the Heme in Cytochrome c, Facilitating Electron Transfer.

Electron transport through biomolecules and in biological transport networks is of great importance to bioenergetics and biocatalysis. More generally, it is of crucial importance to understand how the pathways that connect buried metallocofactors to other cofactors, and to protein surfaces, affect the biological chemistry of metalloproteins. In terms of electron transfer (ET), the strongest coupling pathways usually comprise covalent and hydrogen bonded networks, with a limited number of through-space contacts. Herein, we set out to determine the relative roles of hydrogen bonds involved in ET via an established heme-to-surface tunneling pathway in cytochrome (cyt) c (i.e., heme-W59-D60-E61-N62). A series of cyt c variants were produced where a ruthenium tris(diimine) photooxidant was placed at position 62 via covalent modification of the N62C residue. Surprisingly, variants where the H-bonding residues W59 and D60 were replaced (i.e., W59F and D60A) showed no change in ET rate from the ferrous heme to Ru(III). In contrast, changing the composition of an alternative tunneling pathway (i.e., heme-M64-N63-C62) with the M64L substitution shows a factor of 2 decrease in the rate of heme-to-Ru ET. This pathway involves a through-space tunneling step between the heme and M64 residue, and such steps are usually disfavored. To rationalize why the heme-M64-N63-C62 is preferred, molecular dynamics (MD) simulations and Pathways analysis were employed. These simulations show that the change in heme-Ru ET rates is attributed to different conformations with compressed donor-acceptor distances, by ∼2 Å in pathway distance, in the M64-containing protein as compared to the M64L protein. The change in distance is correlated with changes in the electronic coupling that are in accord with the experimentally observed heme-Ru ET rates. Remarkably, the M64L variation at the core of the protein translates to changes in cofactor dynamics at the protein surface. The surface changes identified by MD simulations include dynamic anion-π and dipole-dipole interactions. These interactions influence the strength of tunneling pathways and ET rates by facilitating decreases in through-space tunneling distances in key coupling pathways.

High-Repetition-Rate Transient Absorption Spectroscopy of Respiratory Supercomplexes.

Hemeproteins are frequent subjects for ultrafast transient absorption spectroscopy (TAS) because of biological importance, strong UV-vis absorption, high photostability, and interesting transient dynamics that depend on redox, conformation, and ligand binding. TAS on hemeproteins is usually performed on isolated, purified proteins, though their response is likely to be different in their native molecular environment, which involves the formation of protein complexes and supercomplexes. Recently, we reported a transient absorption microscopy (TAM) experiment which elicited a transient response from hemeproteins in intact biological tissue using a visible-wavelength pump (530 nm) and probe (490 nm). Here, we find that adaptive noise canceling plus resonant galvanometer scanning enables a high-repetition-rate fiber laser source to make redox-sensitive measurements of cytochrome c (Cyt-c). We investigate the origins of the visible-wavelength response of biological tissue through TAS of intact mitochondrial respiratory supercomplexes, separated via gel electrophoresis. We find that each of these high-molecular-weight gel bands yields a TAS response characteristic of cytochrome hemes, implying that the TAS response of intact cells and tissue originates from not just Cyt-c but a mixture of respiratory cytochromes. We also find differences in excited-state lifetime between wild-type (WT) and a tafazzin-deficient (TAZ) mouse model of mitochondrial disease.

Revision of Commonly Accepted Warburg Mechanism of Cancer Development: Redox-Sensitive Mitochondrial Cytochromes in Breast and Brain Cancers by Raman Imaging.

Simple SummaryWe studied oncogenic processes that characterize human breast cancer (infiltrating ductal carcinoma (IDC)) and human brain tumors: glioma, astrocytoma and medulloblastoma, based on the quantification of cytochrome redox status by exploiting the resonance-enhancement effect of Raman scattering. Our results suggest that the mechanisms controlling the electron transport chain might be deregulated in cancers and indicate that electron transport, organized in terms of electronegativity, is inhibited between complex III and cytochrome c for isolated cells in vitro and between cytochrome c and complex IV in brain and breast tissues. The results provide evidence that the extracellular matrix and interactions with cell microenvironment play an important role in the mechanisms controlling the electron transport chain by cytochrome c.We used Raman imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo human brain and breast tissues, surgically resected specimens of human tissues and in vitro human brain cells of normal astrocytes (NHA), astrocytoma (CRL-1718), glioblastoma (U87-MG) and medulloblastoma (Daoy), and human breast cells of normal cells (MCF 10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231) by means of Raman microspectroscopy at 532 nm. We visualized localization of cytochromes by Raman imaging in the major organelles in cancer cells. We demonstrated that the “redox state Raman marker” of the ferric low-spin heme in cytochrome c at 1584 cm−1 can serve as a sensitive indicator of cancer aggressiveness. We compared concentration of reduced cytochrome c and the grade of cancer aggressiveness in cancer tissues and single cells and specific organelles in cells: nucleous, mitochondrium, lipid droplets, cytoplasm and membrane. We found that the concentration of reduced cytochrome c becomes abnormally high in human brain tumors and breast cancers in human tissues. Our results reveal the universality of Raman vibrational characteristics of mitochondrial cytochromes in metabolic regulation in cancers that arise from epithelial breast cells and brain glial cells.

Polarizability of the Active Site in Enzymatic Catalysis: Cytochrome c.

Anisotropic polarizability of the heme in cytochrome c is found to be a major factor in suppressing the activation barrier of protein electron transfer (catalytic effect). Polarizability couples to the electric field of protein and water to enhance fluctuations of the electron-transfer energy gap and the corresponding variance reorganization energy λvar. The reorganization energy observable by kinetic measurements λr = (λSt)2/λvar is composed of λvar and the Stokes-shift reorganization energy λSt. It is lowered compared to the usually reported λSt due to polarizability of the active site leading to λvar > λSt. The coupling of electrostatic protein-water fluctuations to the polarizable active site is accounted for here by empirical valence-bond diagonalization of the active-site Hamiltonian along the simulation trajectory. We show that recent simulations employing this technique, which failed to find the effect of polarizability on electron-transfer kinetics, were erroneous in neglecting the diagonal dipole moments in the Hamiltonian matrix and failing to rotate the electric field produced by the protein-water medium into the molecular frame of the active site. We find that anisotropy of the tensor of polarizability difference in the two oxidation states of the heme matches anisotropy of the second-rank tensor constructed from the electric field at the active site. Exposure of the heme to water from only one side carries significant catalytic function, directly leading to the field anisotropy and the corresponding depression of the activation barrier.

Application of dynamic disorder approach to the temperature dependent non-exponential electron transfer kinetics in Rhodopseudomonas viridis

The kinetics of electron transfer (ET) reaction from the proximal heme (c-559) of tetraheme cytochrome to the special pair of bacteriochlorophyll (P+), oxidized initially, was probed by absorption experiments in the reaction centre from Rhodopseudomonas viridis between temperatures of 295 K and 153 K. The decay of the kinetic curves is clearly non-exponential at the microsecond timescale. This points to the relevance of having possible influence of dynamic disorder on the reaction kinetics. In this work, based on the Sumi–Marcus model of electron transfer we present a theoretical study to rationalize the experimental results by a microscopic model in which the dynamics of protein is described in terms of the anomalous diffusion of a Brownian particle in a harmonic potential well under the action of fractional Gaussian noise. Starting from a non-Markovian diffusion equation supplemented with an exponential sink term that accounts for the electron transfer reaction between the donor and acceptor groups, we calculate the survival probability from the solution of the corresponding diffusion–reaction equation using the Wilemski–Fixman closure approximation. Our model provides excellent fits to the data, nonetheless being in microsecond timescale made it difficult to access through simulations. Importantly, a number of significant improvements have been made to the original work by our model, such as a single expression for the survival probability explains the kinetic profiles at all the temperatures, the initial decay at 153 K and 173 K are reproduced and the Arrhenius relationship for the relaxation times holds well down to the glass transition temperature. Employing the Arrhenius relation for the relaxation times we quantify the average activation energy for the conformational dynamics which is lower than the previous estimate. Our work suggests an alternative interpretation for the observed non-exponential ET kinetics associated with dynamic disorder, otherwise treated earlier with static heterogeneity.

(Keynote) Application of Electrochemical Surface Plasmon Resonance (ESPR) to the Study of Electroactive Microbial Biofilms

Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode interface (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.

5,7,3′,4′-Hydroxy substituted flavonoids reduce the heme of cytochrome c with a range of rate constants

Flavonoids are antioxidants known to be abundant in edible plants. Seven 5,7,3′,4′-tetrahydroxy substituted flavonoids representing each major flavonoid class were used as cytochrome c reductants to systematically investigate the redox role of their C-rings. Additional examples of flavonoids and benzenediols were investigated to better understand the role of the B-ring. Pseudo-first order rate constants (kobs) and apparent bimolecular rate constants (kapp) values were calculated from spectroscopic measurements. Of the seven flavonoids tested, five yielded measurable observed reduction rate constants. Butein (a chalcone) had the highest apparent bimolecular rate constant (kapp), followed by taxifolin (a flavanonol), catechin (a flavanol), eriodictyol (a flavanone), and luteolin (a flavone). Quercetin (a flavonol) and cyanidin (an anthocyanidin), however, reduced cytochrome c but kapp rate constants were unable to be calculated. Neither this trend nor trends in observed rate constants correlated with flavonoid pKa, solvent accessible surface area, polar surface area, reduction potential, antioxidant ability, resonance, or radical scavenging efficiency. Weak correlation, however, was found with degrees of freedom and the number of redox involved electrons. While some cytochrome c reduction rates have been reported, this study is the first to systematically investigate the role of the structure of the flavonoid C-ring across a full set of flavonoids with identical B-rings.

Comparative analysis of plastocyanin–cytochrome f complex formation in higher plants, green algae and cyanobacteria

Mechanisms of the complex formation between plastocyanin and cytochrome f in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species of cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions force plastocyanin molecule close to the heme of cytochrome f. In the subsequent rotation of plastocyanin molecule around the point of electrostatic contact in the vicinity of cytochrome f, copper (Cu) atom approaches cytochrome heme forming a stable configuration where cytochrome f molecule behaves as a rather rigid body without conformational changes. In Nostoc plastocyanin molecule approaches cytochrome f in a different orientation (head-on) where the stabilization of the plastocyanin-cytochrome f complex is accompanied by the conformational changes of the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation of the approaching molecules was not detected, thus indicating that random motions rather than long-range electrostatic interactions are responsible for the proper mutual orientation. We demonstrated that despite the structural similarity of the investigated electron transport proteins in different photosynthetic organisms, the complexity of molecular mechanisms of the complex formation increases in the following sequence: non-heterocystous cyanobacteria-heterocystous cyanobacteria-green algae-flowering plants.

Ligand accessibility to heme cytochrome b5 coordinating sphere and enzymatic activity enhancement upon tyrosine ionization.

Recently, we observed that at extreme alkaline pH, cytochrome b5 (Cb5) acquires a peroxidase-like activity upon formation of a low spin hemichrome associated with a non-native state. A functional characterization of Cb5, in a wide pH range, shows that oxygenase/peroxidase activities are stimulated in alkaline media, and a correlation between tyrosine ionization and the attained enzymatic activities was noticed, associated with an altered heme spin state, when compared to acidic pH values at which the heme group is released. In these conditions, a competitive assay between imidazole binding and Cb5 endogenous heme ligands revealed the appearance of a binding site for this exogenous ligand that promotes a heme group exposure to the solvent upon ligation. Our results shed light on the mechanism behind Cb5 oxygenase/peroxidase activity stimulation in alkaline media and reveal a role of tyrosinate anion enhancing Cb5 enzymatic activities on the distorted protein before maximum protein unfolding.

Geometrical Changes in the Hemes of Bacterial Surface c-Type Cytochromes Reveal Flexibility in Their Binding Affinity with Minerals.

Microbial extracellular electron transport occurs via the physical and electrical association of outer-membrane c-type cytochromes (OM c-Cyts) with extracellular solid surfaces. However, studies investigating the characteristics of cytochrome binding with solid materials have been limited to the use of purified units of OM c-Cyts dissolved in solution, rather than OM c-Cyts in intact cells, because of the lack of a methodology that specifically allows for the monitoring of OM c-Cyts in whole-cells. Here, we utilized circular dichroism (CD) spectroscopy to examine the molecular mechanisms and binding characteristics of the interaction between MtrC, a unit of OM c-Cyts, in whole Shewanella oneidensis MR-1 cells and hematite nanoparticles. The addition of hematite nanoparticles significantly decreased the intensity of the Soret CD peaks, indicating geometrical changes in the hemes in MtrC associated with their physical contact with hematite. The binding affinity of MtrC estimated using CD spectra changed predominantly depending upon the redox state of MtrC and the concentration of the hematite nanoparticles. In contrast, purified MtrC demonstrated a constant binding affinity following a Langmuir isotherm, with a standard Gibbs free energy of -43 kJ mol-1, suggesting that the flexibility in the binding affinity of MtrC with hematite was specific in membrane-bound protein complex conditions. Overall, these findings suggest that the binding affinity as well as the heme geometry of OM c-Cyts are flexibly modulated in the membrane complex associated with microbe-mineral interactions.

Solution structure of the cytochrome P450 reductase–cytochrome c complex determined by neutron scattering

Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved. The function of these complexes requires specificity of the interaction to allow for selective electron transfer but also a fast turnover of the complex, and they are therefore often transient in nature, making them challenging to study. Here, using small-angle neutron scattering with contrast matching with deuterated protein, we report the solution structure of the electron transfer complex between cytochrome P450 reductase (CPR) and its electron transfer partner cytochrome c. This is the first reported solution structure of a complex between CPR and an electron transfer partner. The structure shows that the interprotein interface includes residues from both the FMN- and FAD-binding domains of CPR. In addition, the FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of CPR. In summary, our results reveal key details of the CPR catalytic mechanism, including interactions of two domains of the reductase with cytochrome c and motions of these domains relative to one another. These findings shed light on interprotein electron transfer in this system and illustrate a powerful approach for studying solution structures of protein–protein complexes.

The Nature and Reactivity of Ferryl Heme in Compounds I and II.

Aerobic organisms have evolved to activate oxygen from the atmosphere, which allows them to catalyze the oxidation of different kinds of substrates. This activation of oxygen is achieved by a metal center (usually iron or copper) buried within a metalloprotein. In the case of iron-containing heme enzymes, the activation of oxygen is achieved by formation of transient iron-oxo (ferryl) intermediates; these intermediates are called Compound I and Compound II. The Compound I and II intermediates were first discovered in the 1930s in horseradish peroxidase, and it is now known that these same species are used across the family of heme enzymes, which include all of the peroxidases, the heme catalases, the P450s, cytochrome c oxidase, and NO synthase. Many years have passed since the first observations, but establishing the chemical nature of these transient ferryl species remains a fundamental question that is relevant to the reactivity, and therefore the usefulness, of these species in biology. This Account summarizes experiments that were conceived and conducted at Leicester and presents our ideas on the chemical nature, stability, and reactivity of these ferryl heme species. We begin by briefly summarizing the early milestones in the field, from the 1940s and 1950s. We present comparisons between the nature and reactivity of the ferryl species in horseradish peroxidase, cytochrome c peroxidase, and ascorbate peroxidase; and we consider different modes of electron delivery to ferryl heme, from different substrates in different peroxidases. We address the question of whether the ferryl heme is best formulated as an (unprotonated) FeIV═O or as a (protonated) FeIV-OH species. A range of spectroscopic approaches (EXAFS, resonance Raman, Mossbauer, and EPR) have been used over many decades to examine this question, and in the last ten years, X-ray crystallography has also been employed. We describe how information from all of these studies has blended together to create an overall picture, and how the recent application of neutron crystallography has directly identified protonation states and has helped to clarify the precise nature of the ferryl heme in cytochrome c peroxidase and ascorbate peroxidase. We draw comparisons between the Compound I and Compound II species that we have observed in peroxidases with those found in other heme systems, notably the P450s, highlighting possible commonality across these heme ferryl systems. The identification of proton locations from neutron structures of these ferryl species opens the door for understanding the proton translocations that need to occur during O-O bond cleavage.

Application of electrochemical surface plasmon resonance (ESPR) to the study of electroactive microbial biofilms.

Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.

59 - Peroxide-Regulated Heme Transfer: A Novel Redox Signaling Mechanism

Using high-performance mass spectrometry (MS), we have uncovered an unprecedented mechanism of redox signaling by H2O2. Remarkably, many cycles of sequential binding and reduction of peroxide can occur in vitro at the heme of cytochrome c peroxidase (Ccp1) in the absence of its reducing substrate, cytochrome c. MS reveals that ~20 oxygen atoms are incorporated into Ccp1, predominantly at methionine and tryptophan residues. Additionally, extensive intramolecular dityrosine formation involving neighboring tyrosines (Y36/Y39, Y36/Y42 and Y67/Y71) was uncovered on MS sequencing of the crosslinked peptides. A total of 24 residues are oxidized by repeated hole-hopping from the heme in Ccp1 treated with a 10-fold excess of H2O2. Oxidation of the catalytic distal H52 shuts down H2O2 reduction but not until the proximal heme ligand (H175) is oxidized, which labilizes the heme. Notably, no irreversible heme modification is detected by MS, indicating that Ccp1 preserves the integrity of its heme by directing the oxidizing equivalents from H2O2 onto its polypeptide. These properties of Ccp1 in vitro are consistent with our observation that it acts as a heme chaperone for apoCta1, a heme-dependent catalase found in yeast mitochondria. When levels of H2O2 rise during the cell’s switch to respiratory metabolism, maturation of Cta1, as monitored by the rise in its catalase activity, coincides with apoCcp1 exit from mitochrondria (1). MS analysis reveals that extramitochondrial Ccp1 is ~80% oxidized at H175, which confirms that its heme has been labilized by mitochondrial H2O2 to trigger heme transfer to apoCta1. This sequence of events constitutes the first example of H2O2 regulating its own intracellular concentration by turning on catalase activity post-translationally via heme transfer. M. Kathiresan, D. Martins and A.M. English Proc. Nat. Acad. Sci., USA 2014, 111, 17468–17473.

Cytochrome c and c1 heme lyases are essential in Plasmodium berghei

Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc1hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc1hl are both essential and thus may be antimalarial targets.