Mitochondrial disease-related mutations at the cytochrome b-iron-sulfur protein (ISP) interface : molecular effects on the large-scale motion of ISP and superoxide generation studied in Rhodobacter capsulatus cytochrome bc_{1}

AbstractOne of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron–sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c1 of cytochrome c1. Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations — G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al., J. Biol. Chem. 290 (2015) 23781-23792]. This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking “semireverse” electron transfer from heme bL to quinone. Here we examine another mutation from that group — G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that “pushing” ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the “pushing” effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS.This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi

Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc(1)

Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc(1), one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH(2)) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Q(i) site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Q(i)-site mediated oxidation of heme b(H), reverse reduction of heme b(H) by quinol and heme b(H)/Q(i) semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH(2) triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license.Peer reviewe

Mitochondrial disease-related mutation G167P in cytochrome b of Rhodobacter capsulatus cytochrome bc_{1} (S151P in human) affects the equilibrium distribution of 2Fe-2S cluster and generation of superoxide

Cytochrome bc(1) is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Q(o) site in cytochrome b with cytochrome c(1). The Q(o) site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Q(o) site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Q(o) site. This renders cytochrome bc(1) non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Q(o) site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc(1) to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which “semireverse” electron transfer back to the Q(o) site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P

Tuning of hemes b equilibrium redox potential is not required for cross-membrane electron transfer

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔE_{m_}b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc1 in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔE_{m_}b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔE_{m_}b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc1 can accommodate large changes in ΔE_{m_}b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔE_{m_}b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential

Atomistic determinants of co-enzyme Q reduction at the Q(i)-site of the cytochrome bc(1) complex

The cytochrome (cyt) bc(1) complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme's substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Q(i)-site of the cyt bc(1) complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Q(i)-site to facilitate binding of half-protonated semiquinone - a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Q(i)-site.Peer reviewe

EPR signals of tumors and spleens from tumor-bearing BALB/cA-nude mice with human lung adenocarcinoma, after PDT treatment

Studies of the nitric oxide production during PDT - inducted antitumor response have been conducted for last 10 years. However there are no clear-cut studies of NO production in vivo in human tumors. In this paper we studied the EPR signals of HbNO complexes from human lung adenocarcinoma in murine model.The major purpose of this study was to detect the EPR triplet signals of nitrosohemoglobin (HbNO) in tumors and spleen samples from tumor-bearing mice previously treated with photodynamic therapy, using ZnPheide as a photosensitizer. The EPR measurements taken on X-band frequency (9.5 GHz) at liquid nitrogen temperature demonstrated HbNO triplet signals in tumor samples, strongly dependent on the state of tumor growth. Only signals of free radical paramagnetic centers have been recorded in the spleen samples.Possible correlation between changes of HbNO signals and photodynamic therapy procedure has also been investigated. However, no correlation between complexity and intensity of HbNO signals and PDT therapy procedure has been demonstrated, as a result of qualitative and quantitative analyses of EPR spectra of tumors.W niniejszej pracy przedstawiono analizę sygnałów EPR guzów ludzkich gruczolakoraków płuca (komórki linii A549) rosnących u myszy bezgrasiczych, w zależności od terapii PDT zastosowanej u tychże myszy.Przeprowadzono pomiary spektroskopii EPR fali ciągłej w paśmie częstotliwości X (9,5 GHz), w temperaturze ciekłego azotu. Dokonywano pomiarów próbek guzów oraz śledzion, pochodzących od myszy poddanych uprzednio terapii fotodynamicznej z zastosowaniem ZnPheide jako fotouczulacza. Na podstawie otrzymanych widm EPR wykonano analizę sygnałów trypletowych pochodzących od kompleksów nitrozylowych z hemoproteinami, w zależności od przeprowadzonej terapii PDT. Wyniki tej analizy wykazały możliwość rejestracji sygnałów trypletowych w badanych próbkach guzów. Próbki śledzion nie charakteryzowały się podobnymi sygnałami, rejestrowano w nich jedynie wolnorodnikowe centra paramagnetyczne. Zmierzone sygnały trypletowe wykazują pewne podobieństwa, w zależności od cech morfologicznych guzów, i prawdopodobnie mogą być wyznacznikiem rozwoju odpowiedzi przeciwnowotworowej organizmu gospodarza. Wykonana analiza widm EPR nie pozwala na jednoznaczne określenie korelacji pomiędzy sygnałami pochodzącymi od kompleksów HbNO a zastosowaną terapią PDT

Identification of hydrogen bonding network for proton transfer at the quinol oxidation site of Rhodobacter capsulatus cytochrome bc1bc_{1}

Cytochrome $bc_{1}$ catalyzes electron transfer from quinol ($QH_{2}$) to cytochrome c in reactions coupled to proton translocation across the energy-conserving membrane. Energetic efficiency of the catalytic cycle is secured by a two-electron, two-proton bifurcation reaction leading to oxidation of $QH_{2}$ and reduction of the Rieske cluster and heme $QH_{2}$. The proton paths associated with this reaction remain elusive. Here we used site-directed mutagenesis and quantum mechanical (QM) calculations to analyze the contribution of protonable side chains located at the heme $QH_{2}$ side of the quinol oxidation site ($Q_{o}$) in Rhodobacter capsulatus cytochrome $bc_{1}$. We observe that the proton path is effectively switched off when H276 and E295 are simultaneously mutated to the non-protonable residues in the H276F/E295V double mutant. The two single mutants, H276F or E295V, are less efficient, but still transfer protons at functionally-relevant rates. Natural selection exposed two single mutations, N279S and M154T, that restored the functional proton transfers in H276F/E295V. QM calculations indicated that H276F/E295V traps the side chain of Y147 in a position distant from $QH_{2}$, while either N279S or M154T induce local changes releasing Y147 from that position. This shortens the distance between the protonable groups of Y147 and D278 and/or increases mobility of the Y147 side chain, which makes Y147 efficient in transferring protons from $QH_{2}$ toward D278 in H276F/E295V. Overall, our study identified an extended hydrogen bonding network, build up by E295, H276, D278 and Y147, involved in efficient removal of the proton from $QH_{2}$ at the heme $b_{L}$ side of $Q_{o}$

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial hemecontaining protein maquettes.Peer reviewe