What is the difference between cytochrome c and cytochrome p450

The ability of both flavin domains to move relatively back and forth from each other is thus a prerequisite for the FMN domain to interact with its physiological acceptors. However, the structural determinants that guide this conformational transition between open and closed forms, a process demonstrated to be essential for gating the interflavin electron transfer in CPR and thus to its redox partners, are still not fully identified.

It has long been known that electron transfer from CPR to cytochromes P displays a strong ionic strength dependency which demonstrated that the electron transfer complex between the CPR and a P is, beside hydrophobic interactions, strongly based on charge pair interactions Tamburini and Schenkman, ; Nadler and Strobel, , ; Davydov et al.

Most of these studies evidenced that ionic interactions between CPR and its redox partners were the major determinants of the salt effects on Ps activities and are reviewed in Hlavica et al. However, we also demonstrated that the ionic strength effect on cytochrome c reduction by the soluble form of human CPR depends on the conformational equilibrium between the locked and unlocked states Frances et al.

From the comparative studies of the open conformation seen in the chimeric yeast-human CPR, two residues in the hinge, G and S, were proposed to be important molecular determinants for the large conformational changes Aigrain et al.

S and R were also found to display large chemical shifts during the change in the conformational equilibrium between the locked and unlocked states Frances et al.

Additionally, the hinge segment was also found to be directly controlling electron transfer to cytochrome c from the reductase domain of nNOS Haque et al. In this work we have targeted four specific residues of the hinge segment indicated above, for site-directed mutagenesis to test their potential role in the conformational equilibrium of human CPR. By analyzing the salt-dependent changes of the cytochrome c reductase activity in the context of both soluble and membrane-bound forms of CPR, we propose several hypotheses on the role played by these residues on the transition between the locked to the unlocked state, and thus in the electron transfer mechanism of CPR.

Louis, MO, United States. Bacto yeast extract was obtained from Formedium Norwich, England. A polyclonal antibody from rabbit serum raised against recombinant human CPR obtained from Genetex Irvine, CA, United States was used for immunodetection of the membrane-bound CPR, while the respective antibody used the in the case of the soluble form of the enzyme was obtained from Thermofisher Scientific Waltham, MA, United States.

DNA sequencing confirmed the absence of any undesired mutations. The constructed plasmids were transformed into competent Escherichia coli BL21 DE3 cells for expression. POR cDNA of plasmids was fully sequenced to confirm the introductions of the designed mutations and the absence of any undesired mutations. CPR mutants were expressed in E. Cell lysis was achieved by 4 cycles of sonication 30 s of burst followed by intervals of 2 min for cooling in buffer A containing a protease inhibitor cocktail aprotinin 0.

Imidazole was eliminated from the eluted fraction by sequential washes in buffer A using a Vivaspin centrifugal concentrator Sartorius, Goettingen, Germany. Expression of the full-length membrane bound CPR mutants was obtained in BTC bacteria and membrane fractions of the different strains were prepared and characterized for protein content as described previously Marohnic et al. CPR content of membrane fractions was quantified by immunodetection against a standard curve of purified human, full-length WT CPR, using polyclonal rabbit anti-CPR primary antibody and biotin-goat anti-rabbit antibody in combination with the fluorescent streptavidin conjugate WesternDot Western Blot Kit; Invitrogen.

With ferricyanide, assays were performed in the same conditions as described above, but in the presence of 1 mM ferricyanide.

Initial experiments were performed with soluble CPR. The reaction was initiated by adding CPR. A set of preliminary rate assays were performed in a microtiter plate format to optimize linearity of the reaction traces for the mutants, with conditions close to the traditional cuvette approach. A first set of kinetic measurements was performed in triplicate using the two cytochrome c concentrations indicated above. A second set of triplicate experiments was repeated with another batch of purified CPR.

CPR samples soluble forms were diluted up to concentrations giving linear velocity traces to compensate for lower or increased velocities, see Supplementary Table S1. Each k obs value is the average of six measurements: one triplicate using a first batch of enzyme and a second triplicate, using a second batch of purified CPR. The conditions of the microplate rate assay were then verified for the membrane-bound CPRs, to ensure linearity of the reaction traces.

Finally, the membrane bound mutants were assayed with CPR well-concentrations, proportionally diluted as those determined for the soluble forms see Supplementary Table S1. Control experiments with E. In order to further understand the role of the ionic strength in electron transfer from CPR to acceptors such as cytochrome c and discriminate between the two major hypotheses salt-dependent conformational equilibrium or electrostatic interactions with the substrates , we analyzed and compared the ionic strength dependency of CPR toward DCPIP and ferricyanide with that of cytochrome c.

DCPIP and ferricyanide have unique characteristics in term of charge and electron receiving capabilities redox potential. Variation of k obs in function of the ionic strength. For cytochrome c , the concentration of NaCl giving the maximal k obs is around mM, a value quite comparable to the one measured in our previous study Frances et al. This difference might be attributed to the fact that DCPIP is an obligate two electrons hydride acceptor, which may have a different overall electron transfer scheme compared to cytochrome c.

The ionic strength dependent variation of k obs measured for the reduction of DCPIP and cytochrome c by soluble human CPR display both a bell-shaped profile. This result thus strengthens the hypothesis that the ionic strength dependency of electron flow from CPR to acceptors is mainly determined by the conformational equilibrium between the locked and unlocked states of CPR and only in a minor manner by electrostatic interactions between the FMN domain and the acceptor.

We also analyzed the reduction of ferricyanide, another non-natural substrate. Interestingly, the salt-dependent k obs profile is totally different from the ones seen with cytochrome c or DCPIP, being composed of two straight lines crossing at a NaCl concentration of around mM. Therefore, the ferricyanide reduction, which mainly occurs at the FAD cofactor Vermilion et al.

Interestingly, the inflection occurs around a salt concentration quite similar to the one yielding the maximum k obs with cytochrome c. However, we do not have any coherent explanation for the peculiar salt profile observed with this substrate. Based on these latter results, we decided to use cytochrome c reduction as an appropriate reporter to probe salt-mediated conformational equilibrium changes in electron transfer of specific CPR mutants.

The hinge segment is defined by a stretch of 14—15 amino acids that does not display any particularly defined secondary structure. These two residues were therefore mutated into prolines in order to test if and how geometrical constraints modify the conformational equilibrium of CPR.

Finally, as the physiological form of CPR is membrane-bound and the membrane anchoring segment may have a profound influence on the equilibrium between locked and unlocked states or the open-closed exchange rates, we analyzed the effects of these mutations in both the soluble and membrane bound context.

Both views provide the rationale for the design of our mutants: G corresponds to the position where the hinge is tilted in the open conformation compared to the closed conformation Figure 2A ; S side chain orientation is different between the two forms Figure 2A ; I and R show an interesting proximity and potential interaction with the connecting domain Figures 2A,B. Positions of the studied mutations in CPR. Mutated residues are displayed as sticks of either green or magenta colors according to the structures.

A Both structures were aligned onto their FMN domains. Biological duplication of the entire expression and purification stages was performed to ensure repeatability. Some discrete bands, at lower molecular weights compared to WT CPR were detected either with Coomassie staining or by western blot analysis see Supplementary Figure S1 , indicating some minor degradation of the purified enzymes.

All CPR mutants presented the same absorbance spectra as the WT soluble enzyme, with a maximum of absorption at nm, indicating no or minor changes in the chemical surrounding of both flavins. Last, all mutants were obtained in the semiquinone form, like the WT CPR, indicating no major changes in the absolute redox potential values of the flavins in the mutant forms.

These results emphasize that the introduced mutations did not apparently modify the surroundings of the flavins. The eight different mutants were introduced in the E. No expression problems were encountered, except for mutant GP for which the level of expression was substantially lower than for the other mutants. Immunodetection of CPR in isolated microsomes showed only trace amounts of the GP mutant protein, not sufficient for subsequent analysis.

We therefore developed and optimized a well plate format assay to follow cytochrome c reduction. Optimal conditions of the microplate rate assay were determined to ensure the linearity of the reaction traces. CPR concentrations in the tests were also optimized to obtain linear traces for each mutant both for the soluble and membrane bound forms in order to compensate for lower or higher activities see Supplementary Table S1.

We verified that salt profiles were equivalent between the tests performed in the cuvette and in the well plate format data not shown. Variation of the k obs for cytochrome c reduction in function of the ionic strength for the WT and mutants of the soluble forms of human CPR. Cytochrome c reduction assays were performed in triplicate in 96 well microtiter plates as described in the Material and Methods section.

A Salt dependent profile of the k obs with the WT and the mutants. Symbols depicting the different mutants are included in the panel.

B Maximal k obs values were extracted from panel A and plotted as a function of the salt concentration. Symbols depicting the different mutants are identical as the ones in A. As mentioned earlier, the ionic strength dependency of cytochrome c reduction by CPR can be partly considered as a direct measure of the proportion of the locked vs. We therefore performed a full analysis of the ionic strength dependency of cytochrome c reduction for all single and double mutants Figure 3.

Figure 3A depicts the 8 different ionic strength profiles obtained with the various mutants compared with the WT one. All curves of the k obs vs. With the exception of the SP mutant, all ionic strength profiles are shifted to the left lower NaCl concentrations. Moreover, nearly all of them except SP and IA have lower maximal k obs values.

The redox potentials of both flavins and cytochrome c are known to depend to some extent on the ionic strength. While for cytochrome c these changes are known and relatively small Gopal et al. Based on our observation that the microenvironment of flavins is probably not significantly altered between mutants and WT CPR equal absorption spectra we have based our working hypotheses on the assumption that the redox potential of the flavins are not functionally different between the mutants and the WT soluble or membrane-bound forms of CPR.

From Figure 3A , maximal k obs values were extracted and used to generate a graph comparing the salt concentration at which the maximum k obs occur and the maximal k obs value itself Figure 3B. The case of SP and IA is interesting. Both have approximately the same k obs slightly higher than the WT for SP.

Another example is the situation for IA, GP and IP, for which the optimal ionic strengths are identical yet the three mutants have different maximal k obs values. Hence these data seem to indicate a separation between the efficiency of cytochrome c reduction and the conformational equilibrium of CPR represented by the salt concentration at which the maximal k obs occurs. However, globally, when the profiles are shifted to lower ionic strength the maximal k obs values are lower.

The effects on the conformational equilibrium are interesting and merit further analysis. With the exception of SP, all mutants either favored the unlocked state or destabilized the locked state. The introduction of a proline residue GP, IP and RP probably imposes strong constraints on the hinge and may therefore stabilize discrete conformations of the unlocked state.

The removal of the positive charge of the arginine residue at position RA may loosen the potential interactions between the hinge and the connecting domain and also destabilize the locked state. Lower maximal k obs values are probably associated to some decrease in the rate constants of one or several electron transfer steps. We already demonstrated that conformational exchange occurs at a much faster rate than electron transfer Frances et al.

This also means that the locked state might actually have several closed conformations, not all of them productive for inter-flavin electron transfer, a feature that has already been described Hay et al. This is a preview of subscription content, log in to check access. Hydrophobic domain, hydrophilic domain, and connecting region. J Biol Chem — PubMed Google Scholar.

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J Biol Chem — Google Scholar. Gekko K and Timasheff SN Mechanism of protein stabilization by glycerol preferential hydration in glycerol-water mixtures. Biochemistry — Google Scholar. Hazzard JT,McLendon G, Cusanovich MA and Tollin G Formation of electrostatically stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase.

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Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species ROS , leading to an inefficient reaction. The ROS produced was not influenced by different physicochemical conditions ionic strength, pH, temperature.

Both Ct CPR structures exhibited the closed conformation. In vivo , CPRs also reduce other proteins such as cytochrome b 5 , heme oxygenase, and the fatty acid elongation system; in vitro , it has been shown to transfer electrons to non-physiological acceptors such as cytochrome c , ferricyanide, and different drugs 2. The structure is well conserved among the different kingdoms, possessing an N -terminal FMN-binding domain that is connected to a C -terminal FAD-binding domain via a linker region.

The proposed mechanism is that during inter-flavin electron transfer, the enzyme is in a compact, closed conformation which facilitates the electron flow between the prosthetic groups. However, this conformation is not ideal for interaction with protein acceptors and therefore CPR undergoes conformational changes to expose the reduced FMN for interaction with CYPs 5.

Despite the different approaches, the overall mechanism and details of the CPR interaction with its redox acceptors remain elusive and further analysis is necessary to completely understand the dynamics of electron shuttle to the CYPs. ROS result in loss of electrons and leads to an inefficient reaction, oxidative damage and ultimately inactivates the enzymes In this study, we investigated ROS production by C.

We analysed the production of H 2 O 2 and superoxide during Ct CPR activity, as well as the influence of different physicochemical parameters on uncoupling. Both structures revealed Ct CPR in the closed conformation. The overall structures are similar to those previously reported for mammalian and yeast proteins 21 , 22 , We discuss the differences and similarities between the different CPRs and review the regulatory function of inter-domain ionic interactions.

The genes are alleles with high identity between the protein sequences and have similar activity rates. In this work, we expressed the Ct CPR coded by cpr-b.

The N -terminal domain of eukaryotic CPR is a highly hydrophobic sequence that acts as a membrane anchor. One strategy for the solubilization of membrane-associated proteins is the deletion of this hydrophobic region, which generally improves heterologous expression in Escherichia coli Protein purification was a three-step procedure involving low- and high-resolution anion-exchange chromatography, followed by size-exclusion chromatography, to achieve a high level of purity Figs.

Based on the average concentration of these peaks, we estimated a yield of 0. For titration of the flavin groups, increasing concentration of NADPH was added and absorbance spectra were recorded after reaching equilibrium Fig. Further addition of NADPH produced little change in the semiquinone peak and a decrease in absorbance at lower wavelengths, which represents the transition into different states of the flavin groups.

It is well established that the directional flow of electrons during CPR catalysis is initiated by a hydride transfer from NADPH to the FAD group, where after direct inter-flavin electron transfer takes place. Finally, the electrons are shuttled to the external acceptor from the FMN group. However, no consensus has been reached concerning the reducing cycle and the active form of FMN that donates electrons to the acceptor.

Saturation curves for both substrates followed Michaelis-Menten behaviour Fig. Although these molecules are not physiological substrates of the enzyme, they are commonly used to evaluate CPR activity due to the simplicity of the assays.

FeCN, and other small molecules, only require one electron, which can be donated directly from FAD Cytochrome c reduction is proposed as an analogue to natural substrates 37 , but being a smaller protein, it does not share the same reduction mechanism or binding site as the CYPs 38 , More recently, it has been proposed that cytochrome c can receive electrons without binding to the reductase GC-MS analysis of the biotransformations with dodecanoic acid showed the expected production of hydroxydodecanoic acid Fig.

The CPR supplies electrons to a wide variety of microsomal CYPs that are involved in important metabolic functions in vivo and are highly valuable for medical and technological applications During electron transfer, uncoupling can produce ROS species through the non-productive activation of oxygen, generating H 2 O 2 and superoxide anions The mechanism and factors that influence this phenomenon are not completely understood, but it is known that the coupling efficiency varies depending on the P system used 19 , as both CYP and CPR can contribute to ROS formation.

We used a previously described spectrophotometric method to quantify the production of both H 2 O 2 and superoxide, by the addition of superoxide dismutase Relevant controls confirmed that the heating of the samples did not affect ROS production Fig. H 2 O 2 was the major product of uncoupling, since the addition of SOD only slightly increased the total amount of H 2 O 2 detected Fig. Similar results were obtained for C. We observed similar levels of NADPH consumption in reactions with cytochrome c , although the electrons were mainly used to reduce the acceptor and ROS production was on average 3 times lower compared with reactions without cytochrome c Fig.

Since it has previously been reported that different factors can affect the coupling efficiency of the system 45 , we investigated the effect of different physicochemical conditions on Ct CPR activity and their influence on ROS production.

Uncoupling of Ct CPR under different conditions. We analysed the effect of ionic strength by performing the reactions with different concentrations of NaCl. NADPH consumption rates increased with ionic strength up to 1. Previous work 9 , 11 , 48 has shown that higher ionic strength favours the open conformation of the CPR, increasing the catalytic efficiency towards cytochrome c.

SANS data showed a considerable increase in the flexibility of the enzyme, suggesting that the CPR might be partially unfolded under high ionic strength In addition, we examined the impact of pH and temperature on uncoupling. These findings correspond with the idea that an open state favours the activity of the enzyme. Interestingly, the percentage of uncoupling was not influenced by different conditions Table S3.

Both structures were obtained in the closed conformation Fig. More recently, a semi-open structure has been solved for the CPR from Arabidopsis thaliana Crystal structure of Ct CPR.

Even though only 22 residues were truncated at the N-terminus, density was only observed from residue 45, suggesting a relatively long flexible N -terminal region. The FMN-binding topology is conserved among the different CPRs, with the isoalloxazine ring hydrogen-bonded to backbone atoms in addition to some water-mediated hydrogen bonds. The phosphate group is similarly bound but also contacts directly to the sidechains of Ser65, Thr67 and Thr69 in addition to a more extensive water bridge network Figs.

S9 , the adenosine moiety is buried, sandwiched between Pro and Val, and hydrogen-bonded via N6A to the side chain oxygen of Asn and the main chain carbonyl oxygen of Phe Fig.

The Arg electrostatically stabilizing the FAD through interaction with the diphosphate is also conserved. Apart from the direct hydrogen bond formed with the side chain of Ser, the isoalloxazine ring of FAD is mostly hydrogen-bonded to main-chain carbonyl and amine groups Fig.

This residue is conserved among CPRs and has been implicated in the regulation of electron transfer 50 , As the tryptophan sterically prevents the binding of the nicotinamide part of NADPH, movement is required for hydride transfer to occur. FAD and C -terminal Trp are also displayed. These ionic interactions or locational equivalents in other solved CPR structures are mostly solvent-exposed and highly conserved among the different CPRs.

These interactions will, however, be significantly weakened with increased salt concentrations such as that found under physiological conditions. The two domains are linked via a flexible hinge region, allowing the FMN domain to rotate away from the FAD domain for electron transfer to its physiological partner proteins.

This conformational freedom has to be countered so that once the enzyme returns to the closed conformation, the correct interfacial domain interaction is achieved.

The conserved salt-bridges could possibly direct the domain associations for successful and productive orientations by reducing the potential relative orientations. It has also been shown that the hinge is important for the conformational equilibrium in hCPR and therefore the reaction rate of the enzyme However, comparison of the amino acid sequences from CPRs from different organisms revealed that the hinge differs significantly in CPRs from different species and that these specific ionic interactions are not conserved.

The differences in salt bridges formed in mammalian and yeast enzymes might influence the ability of the yeast-human chimeric protein to stabilize the closed conformation, promoting the open conformation which would have an effect on its activity These results suggest the importance of the ionic interactions in constraining the flexibility of the CPR to optimize proper electron transfer. In this study, we describe the functional expression of the CPR from C.

Molecular insights gained into the interfacial domain interactions suggest that specific conserved ionic interactions contribute to the flexibility and conformational equilibrium of the protein, which in turn, would affect reaction rates. The cpr-b gene from C. The sequence was optimized for E. The open reading frame of cyp52a21 from C. For expression of CYP52A21, media was supplemented with 0. The desalted protein was subjected to a second round of anion exchange chromatography.

The eluted enzyme was pooled and concentrated as described above. For uncoupling determination, hydrogen peroxide production was quantified using Ampiflu TM Red assay 19 in the presence and absence of cytochrome c. Reactions were started with the addition of X-ray diffraction data were collected at Diamond Synchrotron UK on beamline i 0.

Iterative cycles of manual model building in COOT 61 and refinement using refmac 62 were performed and model building progress was monitored by changes in R free and R work values after each refinement cycle.

Porter, T. New insights into the role of cytochrome P reductase POR in microsomal redox biology. Acta Pharm. B 2 , — Iyanagi, T. Hanneman, F. Cytochrome P systems - biological variations of electron transport chains.