Physiological functions of the respiratory
cytochrome bd and the CuA nitric oxide reductase
(CuANor)
PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus prof. ir. K. C. A. M. Luyben; voorzitter van het College voor Promoties, in het openbaar te verdedigen op woensdag 25 november 2015 om 10:00 uur doorSinan AL-ATTAR
Master of Science, Technische Universiteit Delft, Nederland geboren te Basrah, IrakThis dissertation has been approved by the promotor: Prof. dr. S. de Vries† Composition of the doctoral committee: Rector Magnificus Chairman Prof. dr. S. de Vries† Delft University of Technology Independent members: Prof. dr. ir. M. C. M. van Loosdrecht Delft University of Technology Prof. dr. P. Moënne-Loccoz Oregon Health & Science University Prof. dr. W. J. H. van Berkel Wageningen University Prof. dr. P. D. E. M. Verhaert Delft University of Technology Prof. dr. U. Hanefeld Delft University of Technology Dr. P. A. S. Daran-Lapujade Delft University of Technology Copyright © 2015 Sinan Al-Attar
To my family In the memory of my teacher, professor Simon de Vries
Contents
Samenvatting (Dutch) 1 Summary 3 Chapter 1 General introduction 5 Chapter 2 Energy transduction by respiratory metallo-enzymes: From molecular mechanism to cell physiology 23 Chapter 3 Cytochrome bd is a quinol peroxidase proposed to protect prokaryotes from exogenous hydrogen peroxide 69 Chapter 4 An electrogenic nitric oxide reductase 91 Chapter 5 Expression of Bacillus azotoformans c-cytochromes and synthesis of phenazine ethosulfate sulfonate 119 Chapter 6 Conclusions and outlook 127 Acknowledgements 131 Curriculum Vitae and list of publications 1331
Samenvatting
Celmembranen vormen de bindingsplaats voor een verscheidenheid aan eiwitten die onmisbaar zijn voor de cel. De ademhalingsketen in de cel, die voornamelijk bestaat uit membraangebonden eiwitten, zorgt er voor dat de elektronen afkomstig van voeding zich als een elektrische stroom verplaatsten van redoxkoppels met een lage naar die met een hoge redox midpointpotentiaal. In dit proces van opeenvolgende stappen van elektronenoverdracht, wordt devrijkomende chemische energie omgezet in een transmembraan
elektrochemische gradiënt. Deze vorm van potentiële energie wordt naderhand gebruikt voor de productie van ATP. In Hoofdstuk 2 worden de mechanismen besproken die verantwoordelijk zijn voor het creëren van elektrochemische gradiënten.
Cytochroom bd is een terminale oxidase dat alleen voorkomt in de ademhalingsketen van prokaryoten. Het enzym is elektrogeen en zorgt voor de reductie van zuurstof naar water met elektronen afkomstig van quinol. Studies hebben aangetoond dat het ontbreken van een functioneel cytochroom bd in bacteriën leidt tot verhoogde gevoeligheid voor waterstofperoxide (H2O2),
ophoping van H2O2 en verminderde virulentie in pathogene prokaryoten. In dit
onderzoek wordt aangetoond dat cytochroom bd uit Escherichia coli in staat is H2O2 te reduceren tot water via quinol peroxidatie (Hoofdstuk 3). De bevinding
dat cytochroom bd een quiolperoxidase is, verschaft de moleculaire basis voor zijn rol om de bacteriële cel te beschermen tegen exogeen waterstof peroxide afkomstig van fagosomen of van concurrerende bacteriën.
Een ander ademhalingsketenenzym, stikstofmonoxide reductase (Nor), is één van de denitrificatie-enzymen verantwoordelijk voor het reduceren van stikstofmonoxide (NO) naar distikstofoxide (N2O), ook wel lachgas genoemd. De
cytochroom c afhankelijke cNor is een niet-elektrogeen enzym waarbij de energie die vrijkomt bij reductie van NO niet bijdraagt aan de vorming van een elektrochemische gradiënt. Aan de andere kant, de quinol-afhankelijke qNor wordt verondersteld elektrogeen te zijn gebaseerd op de 3D-structuur die recentelijk was opgehelderd. qNor komt vaak voor in niet-denitrificerende organismen en wordt beschouwd as een virulentiefactor van belang voor het detoxificeren van NO geproduceerd in fagosomen. Een derde type Nor is de koper-A-Nor (CuANor) uit Bacillus azotoformans (Hoofdstukken 4 en 5) die
significante homologie vertoont met de cytochroom ba3 oxidase. In dit werk
hebben we met behulp van homologiemodelering (homology modeling) aangetoond dat CuANor de routes voor protonenoverdracht bevat die ook
2
proteoliposomen met CuANor, hebben we aangetoond dat CuANor een
elektrogeen enzym is dat de reductie van NO direct koppelt aan de vorming van een protonelektrochemische gradiënt. Hierdoor, zouden Bacilli door gebruik te maken van CuANor, meer ATP genereren dan denitrificerende organismen die
alleen een cNor bevatten.
Meer inzicht in het samenspel tussen de rol van ademhalingsenzymen in de energiestofwisseling en hun rol in detoxificatie, is van belang bij het ontwerpen van nieuwe targets voor antibiotica tegen humane pathogenen.
3
Summary
Biological membranes form a binding platform for a variety of proteins vital to the cell. The respiratory chain consists mostly of membrane-bound enzymes. These enzymes form a functional chain in which electrons derived from nutrition successively flow from low potential to high potential redox couples. During this process, the chemical energy is transformed to potential energy in the form of a transmembrane electrochemical gradient, which is subsequently stored in the form of ATP. The operation of this electron transfer chain and the mechanisms whereby a transmembrane electrochemical gradient is formed and utilized for ATP production are reviewed (Chapter 2).
Cytochrome bd is a terminal oxidase of the aerobic respiratory pathway that catalyzes the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Reports have shown that disruption of cytochrome bd in bacteria leads to increased sensitivity to hydrogen peroxide (H2O2), accumulation of H2O2
and decreased virulence. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase that reduces hydrogen peroxide to water (Chapter 3). The observation that cytochrome bd is a quinol peroxidase, provides a biochemical basis for its role in detoxification of exogenous peroxide such as encountered in phagosomes or generated by competing bacteria in the natural environment.
Another respiratory enzyme, nitric oxide reductase (Nor), an enzyme of the denitrification pathway, reduces nitric oxide (NO) to nitrous oxide (N2O). The
cytochrome-c-dependent cNor was shown to be non-electrogenic, i.e. unable to conserve energy. Based on its structure, the quinol-dependent qNor, was suggested to be an electrogenic enzyme and is implicated in virulence, as it can detoxify the NO produced in phagocytes. A third type of Nor is the copper-A-Nor (CuANor) from Bacillus azotoformans (Chapters 4 and 5). This enzyme is highly
homologous to the proton-pumping cytochrome ba3 oxidase. Homology modeling
indicated that CuANor contains the proton entry and proton exit pathways
implicated in proton pumping by the cytochrome ba3 oxidase. Using
proteoliposomes, we show here that CuANor is electrogenic and thus couples NO
reduction directly to the formation of a proton-dependent electochemical gradient. By employing CuANor bacilli generate more ATP than organisms that
employ cNOR.
More insight into the interplay between respiration for bioenergetic ends and “respiration” with detoxification as a goal can aid in the design of bacteriocidal drugs to combat human pathogens.
4
5
Chapter 1
General Introduction
6
Aerobic respiration and denitrificationIn the absence of oxygen, organisms can thrive on terminal electron acceptors such as fumarate, CO2, metal ions, sulfate and nitrogenous anions like nitrate or
nitrite [1]. Between 3.2 and 3.5 billion years ago the first photosynthetic organisms that released oxygen into the biosphere appeared on the primordial earth [2]. Although the oxygen tension remained very low for approximately the next billion years due to the oxidation of abundant ferrous iron and sulfide, this event opened a new era in biological evolution enabling organisms to transduce more energy by employing aerobic respiration where oxygen is reduced to water. Aerobic respiration is a multistep process found in all domains of life, which metabolically couples oxygen reduction to the oxidation of reduced organic carbon compounds to CO2.
Compared to the quite simple and usually linear mitochondrial respiratory chains in eukaryotes, the respiratory chains in prokaryotes are complex and branched [1, 3]. Prokaryotes can divert electron flow by expression of specific respiratory modules. This permits them to use many different terminal electron acceptors and also allows for the use of the same terminal electron acceptor but employing different enzymes that can be considered iso-enzymes. This complex network of electron donors, electron acceptors and catalysts provide prokaryotes with great flexibility and allows for delicate adaptation to the environment.
The core of the respiratory chain in prokaryotes consists of trans-membrane enzymes embedded in the cytoplasmic membrane. NADH is produced by glycolysis and the tricarboxylic acid cycle and mediates electrons to the respiratory pathway [4]. NADH dehydrogenase oxidizes NADH and reduces the membrane-soluble electron carrier, quinone [5]. Quinol, the reduced form of quinone, is the substrate for the next complex in the respiratory chain, the cytochrome bc1 complex, which in turn oxidizes the quinol to quinone and
reduces ferric cytochrome c to the ferrous form [6]. Depending on the organism, electron donors other than NADH can be used to replenish the quinone pool among which succinate, formate, glycerolphosphate, pyruvate, lactate and hydrogen [1]. Reduced cytochrome c and quinol function as electron donors to a variety of respiratory enzymes in prokaryotes.
Two independently evolved types of terminal oxidases, cytochrome oxidases (Coxs) and the cytochrome bd-type oxidases, reduce oxygen to water [7]. Coxs are members of the superfamily of the heme-copper oxidases (HCOs), which obtain their electrons from quinols or cytochrome c depending on their type [8-10]. For example, Escherichia coli which, in contrast to Paracoccus denitrificans,
7
lacks a cytochrome bc1 complex and cytochrome c and expresses the
quinol-dependent Cox cytochrome bo3 [11]. The bd-type oxygen reductases (for example
cytochromes bd-I and bd-II in E. coli [12]) constitute an alternative prokaryotic branch for the electrons from quinol to oxygen and can exist side-by-side with the Coxs in the same organism [13]. The free energy liberated during each of the oxidoreduction steps (NADH:quinone, quinol:cytochrome c, quinol:O2 and
cytochrome c:O2) is utilized to create a proton electrochemical gradient over the
membrane which subsequently drives ATP synthesis through the FoF1 ATP
synthase [4]. More details on the aerobic respiratory chain and its bioenergetics are given in the review in Chapter 2.
Besides aerobic respiration, denitrification is a widespread respiration pathway found in many proteobacteria and archaea [14-17]. Organisms that are able to respire on nitrate or nitrite and produce nitrous oxide (N2O) or dinitrogen gas
(N2) are recognized as denitrifiers. Complete denitrification employs four
enzymes: Nar, Nir, Nor and N2Or which successively reduce nitrate (NO3-), nitrite
(NO2-), nitric oxide (NO) and nitrous oxide to nitrogen gas, respectively. The
reducing equivalents are donated by cytochrome c, pseudoazurin or quinol [18].
Denitrification is part of the so-called global nitrogen cycle (Fig. 1), the network of interconversion of nitrogenous compounds. Nitrogen is indispensible as it is a constituent of two main building blocks of life, amino acids and nucleic acids. Gaseous dinitrogen is abundant in the earthly atmosphere but cannot be used in this form as N-source by the majority of organisms. Many microorganisms possess, however, the capacity to capture N2 and reduce it to ammonium using
the enzyme nitrogenase [19] making nitrogen accessible for other life forms. The central processes of the nitrogen cycle carried out by microorganisms are dinitrogen fixation, nitrification, assimilatory nitrate/nitrite reduction, dissimilatory nitrate/nitrite reduction to ammonium (DNRA), anaerobic ammonium oxidation and denitrification [20, 21] (Fig. 1). Dinitrogen fixation produces ammonium by free-living or symbiotic bacteria [22]. However, ammonium can be converted directly to N2 in the presence of nitrite via
anaerobic ammonium oxidation [23] leading just as denitrfication to the loss of mineral nitrogen. Besides the bacterial production of ammonium via dinitrogen fixation and DNRA (Fig. 1), humans lead to the production of significant amounts of ammonium through the synthesis of fertilizers (ammonium nitrate) and the treatment of waste water [24-27]. Fixed nitrogen in the form of ammonium promotes nitrification and leads to the production of nitrate and nitrite, which are converted to N2 mainly by denitrification. The loss of fixed nitrogen via
coupled nitrification/denitrification greatly reduces the efficiency of fertilizers.
8
In Figure 2, a simplified scheme is presented outlining the branched denitrification and aerobic respiratory pathways of Paracoccus denitrificans and Escherichia coli [1]. In this introductory chapter, the focus will be on cytochrome bd and nitric oxide reductase as these two respiratory enzymes constitute the scope of this thesis.
Figure 1 Schematic drawing of the nitrogen cycle (See [14] and [28])
NH
4
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Dinit
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2
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Denitrification
An
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ammonium oxidation
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(%
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Fertilizers
NO
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9
Figure 2 A simplified scheme of the aerobic and denitrification pathways in Escherichia coli and
Paracoccus denitrificans, respectively [1]. Black solid arrows, dashed gray arrows and gray solid arrows
indicate electron transfer steps that take place in P. denitrificans, E. coli and in both, respectively. Successive arrows indicate that more that one enzymatic step is involved. Enzyme names encircled with solid frames indicate enzymes that are electrogenic. Dashed frames indicate enzymes that can be either electrogenic or not, depending on the type. No frame indicates that the enzyme is not electrogenic. Cytochrome bd is abbreviated as bd, cytochrome c as Cc and quinols as QH2. The other abbreviations are defined in the text.
NADH%
dehydrogenases%
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%bd$
Nar%
Nir%
Nor%
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Other%enzymes%
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2"nutrients
10
Cytochrome bd
Cytochrome bd is a membrane-bound terminal oxygen reductase that oxidizes quinol and catalyzes the production of water from oxygen (Eq. 1) [12]. This strictly prokaryotic oxidase is found in many (opportunistic) pathogens [29-38] and contributes to the formation of the proton motive force by consuming protons (chemical protons) from the cytoplasm and electrons from the periplasm without actual proton pumping [13, 39-42]. Protons are taken up at the cytoplasmic (cyto) side of the membrane and electrons from quinol enter at the periplasmic (peri) side (Eq. 1) 2𝑄𝐻!+ 𝑂!+ 4𝐻!"#$! → 2𝑄 + 2𝐻 !𝑂 + 4𝐻!"#$! Eq.1
Cytochrome bd consists of two large subunits designated CydA and CydB and binds a small subunit, CydX. The latter has only recently been shown to associate to cytochrome bd and is needed for its wild type activity [43-46]. See also Chapter 3.
Cytochrome bd carries two b-type hemes and a chlorin (heme d). Heme b558 is a
low-spin hexa-coordinated porphyrin moiety implicated in transferring the electrons from quinol to the oxygen reduction site. The high-spin penta-coordinate hemes b595 and d are proposed to constitute a functional binuclear
site, similar to the binuclear heme-CuB site in Cox where the oxygen chemistry
takes place [47] (Fig. 3). The reduction of oxygen occurs in a concerted 4-electron redox reaction preventing the formation of reactive oxygen species [48]. The bioenergetic efficiency of cytochrome bd is half of that of the oxygen-reducing Coxs, which in addition to consuming chemical protons, also pump protons across the membrane (Eq. 2) [49, 50].
4 𝑒!+ 𝑂
!+ (4 + 𝑛)𝐻!"#$! → 2𝐻!𝑂 + 4𝐻!"#$! + (4 + 𝑛)𝐻!!"#! Eq. 2
Herein n is the number of protons being pumped from the cytoplasm to the periplasm. More details concerning proton pumping are presented in Chapter 2.
It has been shown that mutants carrying disrupted cytochrome bd display high sensitivity to hydrogen peroxide, increased levels of endogenous hydrogen peroxide and decreased virulence [30, 34-37, 45, 51-57]. The biochemical basis for the protective effects of cytochrome bd have so far not been elucidated. Recent studies have suggested that cytochrome bd can function as a catalase [58]. In Chapter 3 the activity of cytochrome bd towards hydrogen peroxide is investigated.
11
Figure 3 Schematic drawing of cytochrome bd embedded in the membrane. CydA is shown on the left
side, CydB in the middle and CydX on the right. The 3D structure of the enzyme is not known, Electrons are donated by quinol and migrate from left to right through heme b558 and further to the binuclear site made up of hemes b595 and d where oxygen reduction takes place. The reaction is electrogenic due to the consumption of protons from the cytoplasmic side of the membrane. The heme centers are drawn as diamonds. Thin dashed arrow indicates electron transfer.
Nitric oxide reductase
Among the intermediates of the denitrification pathway, NO and N2O are of
special interest because of the negative environmental impact of N2O and the
high cytotoxicity of NO. N2O results from incomplete denitrification and is an
ozone scavenger and a notorious greenhouse gas 300 times more potent than CO2
whose emissions have to be controlled [59, 60]. NO is employed by higher eukaryotes as a signaling molecule and is used as a cytotoxin by macrophages to combat infections and tumors [61, 62].
Nors are integral membrane enzymes, which promote the N-N bond formation between two NO molecules consuming two protons and two electrons and producing one N2O (Eq. 3) [14, 63, 64]. 2𝑁𝑂 + 2𝐻!+ 2𝑒!→ 𝑁 !𝑂 + 𝐻!𝑂 Eq. 3 Although Nors lack copper at their active site, they are classified as members of the heme-copper oxidase (HCO) superfamily [65, 66]. HCOs contain 12-14 transmembrane alpha-helices (TMHs) and six conserved histidines, which coordinate the redox centers [67]. Coxs and Nors show cross-reactivity with their respective electron acceptors (O2 and NO) and mutations in Nors showed that
reduction of O2 and NO are affected in a similar fashion [7, 68-77]. Both enzyme
families share a similar catalytic core, which consists of a low-spin heme (b in
4 H
+ cytoplasm periplasm 2"QH2"O
24 H
+ 2 H2O12
Nors and a or b in Coxs) and a binuclear metal center (BNC) where the NO or O2
chemistry takes place [15, 67, 78-80]. The BNC in Nor contains a high-spin heme b3 [80-82] and a non-heme iron designated FeB. In Coxs, FeB is exchanged for CuB
and the heme at the BNC can be of type a3, o3 or b3 [15, 18]. The two Nors that
have so far been crystallized show the presence of a calcium ion that bridges the propionates of hemes b and b3. In addition to these Nors, also cytochrome cbb3
possesses this calcium ion, but not the other members of the HCO superfamily. In terms of evolution, cytochrome cbb3 is the closest relative to the Nors among the
oxidases of the HCO superfamily [83]. Despite the lack of information on the role of the calcium ion in Nors, mutation of one of its ligands shows a detrimental effect on NO reduction proving its functional importance [84, 85]. The reaction mechanism with respect to substrate binding and electron transfer in Coxs is quite well described and widely accepted, although the precise stoichiometry of proton pumping, 0.5 H+/e- for Type B and Type C oxidases or 1 H+/e- for all
oxidases still poses controversy [7, 86, 87]. The precise reaction mechanism of Nors is poorly understood, nonetheless three potential mechanisms are generally recognized: trans-, cis:heme b3- and cis:FeB
-mechanism [16, 18, 64, 79, 80, 82, 88-91]. According to the trans-mechanism, one NO molecule binds to each iron in the BNC forming two mononitrosyls that join to form a hyponitrous intermediate in which the N-N bond has been formed [79, 80, 89]. The other two mechanisms (cis:heme b3 and cis:FeB) involve the successive binding of two NO molecules to
the same iron center [92]. A notable kinetic property of Nor (not found in other HCOs) is substrate inhibition at low micromolar NO concentrations resulting in sigmoidal steady-state kinetics [15, 80, 93, 94].
The reduction of O2 to water is a highly exergonic reaction with a reduction
potential at pH 7 of 0.81 V for the redox couple O2/H2O. A substantial amount of
the free energy of oxygen reduction for example by cytochrome c (<0.3 V) in Coxs is conserved in the form of a proton electrochemical gradient. Although NO reduction is more exergonic (1.2 V) than O2 reduction, experiments with Nor
(cNOR, see below) show that the enzyme does not conserve energy.
Three classes of Nor are acknowledged (Fig. 4): cytochrome c (Cc) oxidizing cNor [95, 96]; quinol-dependent qNor [84, 94] and the copper-A-dependent CuANor
from Bacillus azotoformans that receives electrons from cytochrome c551 [97].
The most extensively studied Nor is the heterodimeric cNor which contains the catalytic subunit, NorB and the membrane-anchored cytochrome c, NorC, serving as the electron entry site. cNor is solely found in denitrifying prokaryotes [14, 98] and is experimentally established to be non-electrogenic [95, 99-102]. cNor from the Gram-negative Pseudomonas aeruginosa, was the first Nor to be crystallized [96]. Computational, mutational and structural analyses of Pseudomonas and
13
Paracoccus cNors (52% sequence identity [100]), suggested that the enzyme takes both the electrons and protons from the periplasm rendering the enzyme non-electrogenic [96, 100, 101, 103] and thus in agreement with the empirical evidence. More recently the crystal structure (2.5 Å) was solved for the quinol-dependent single-subunit qNor (NorZ, also called NorB in other organisms) from the Gram-positive Geobacillus stearothermophilus [84]. The structure revealed the presence of a water-filled channel extending from the cytoplasm to the active site and was proposed as a proton transfer pathway. This suggests that the protons in qNor are consumed from the cytoplasm while the electrons originate from the opposite side of the membrane in contrast to what was found in cNor (Fig. 4) [84, 104]. The electrogenicity of qNor has so far not been experimentally confirmed.
Over decades, the studies on denitrification have mainly focused on Gram-negative organisms [105]. B. azotoformans is a Gram-positive soil bacterium that contains the entire denitrification pathway and is capable of respiring on nitrate, nitrite or NO with N2 as the end product [106]. This organism harbors all the
denitrification enzymes in its cytoplasmic membrane, in contrast to Gram-negative organisms, and is considered an appropriate model organism for the study of denitrification in Gram-positives [93, 107]. It was shown that B. azotoformans can grow on glycerol anaerobically in the presence of nitrate and did not display succinate dehydrogenation activity [107]. Thus nitrate, nitrite and N2
O reduction in B. azotoformans is “fueled” by menaquinol from the rotenone-insensitive NADH dehydrogenase. NO reduction by CuANor is dependent on
quinol:cytochrome c oxidoreductase activity by the b6f complex [107]. CuANor is
uniquely found in bacilli [108] and in contrast to cNor and qNor, the enzyme binds a di-copper cofactor called CuA similar to that found in Coxs [93, 109, 110].
CuA is presumed to be the site where cytochrome c is oxidized just as is Coxs.
CuANor shares high sequence and structural similarities with cytochrome ba3
oxidase from Thermus thermophilus (Chapter 4). It was shown that the enzyme does not accept electrons from horse heart cytochrome c unlike cNor from Paracoccus denitrificans [95, 97]. CuANor is active with the endogenous
cytochrome c551, which has a relatively low midpoint potential (~140 mV) and an
overall charge that is negative [97]. These latter two properties can explain why horse heart cytochrome c was found to be inactive with CuANor.
In Chapter 4, the results are presented which show that CuANor from B.
azotoformans is, so far unique, an electrogenic Nor that consumes protons from the cytoplasmic side of the membrane (Fig. 4).
14
Figure 4 A schematic representation of the three types of Nor showing the origin of protons needed for
the reduction of NO to N2O (see Eq. 3). cNor is a non-electrogenic enzyme because it consumes the protons needed for NO reduction from the periplasmic side. qNor is suggested (dashed arrow) to consume protons from the cytoplasmic side based on its structure while CuANor is shown to be electrogenic by consuming protons from the cytoplasmic side (Chapter 4). Hemes are shown as white squares and the non-heme iron center (FeB) as white circles. Thin dashed arrows indicate electron transfer.
Aim of the thesis
For decades oxidative phosphorylation in mitochondria has served as a paradigm for the bioenergetic process in prokaryotes. At present, all enzymes of the mitochondrial respiratory chain, including the FoF1 ATP synthase have been
crystallized [111-119] and are well studied in terms of their mechanistic features. Since around the 1980s, with the advent of DNA sequencing and molecular cloning techniques, the mitochondrial studies have been complemented with structural and kinetic studies on bacterial enzymes and enzymes from yeast and fungi. Initially these studies focused on the enzymes of the aerobic respiratory chain of P. denitrificans [120-124] because these enzymes were found most similar to those in mitochondria both functionally and in terms of the primary sequence determined from the DNA sequence. However, the bacterial enzymes are less complex than their mitochondrial counterparts containing fewer subunits and are apparently made up only of the catalytic core subunits. Studies with bacterial enzymes also enabled mutational analyses that greatly contributed to our understanding of the function of specific residues in catalysis both in the bacterial and the mitochondrial enzymes [111, 125-134]. The diversity of prokaryotic respiratory chains is enormous, and perhaps much larger than expected. The nitrogen cycle (Fig. 1) may serve as an example containing many more pathways than envisioned in 1997 [14]. In fact, a recent study suggests an additional pathway, the reverse hydroxylamine:ubiquinone reductase pathway (reverse-HURM) with the mutagenic free hydroxylamine as an intermediate [135]. While aerobic respiration and denitrification mainly serve
Cc" 2H+
cNor qNor CuANor
Cc" 2H+ 2H+ CuA QH2% cytoplasm periplasm
15
the bioenergetics of the cell these pathways communicate in various ways with the other metabolic pathways. They affect the redox state of the cell, the ATP/ADP balance, produce toxic intermediates like ROS (superoxide, hydrogenperoxide) and RNS, reactive nitrogen species (NO, NO2-, NH2OH).
Cytochrome bd and Nor are two examples of respiratory enzymes that besides their obvious respiratory function can function in cellular protection against hydrogen peroxide and NO, respectively, two agents used by immune systems to combat bacteria [136-138]. Cytochrome bd is recognized as a virulence factor, but it is unknown how the enzyme confers virulence. We show in Chapter 3 that cytochrome bd has significant quinol peroxidase activity. We propose that this activity accounts for the protective role of cytochrome bd against H2O2 in vivo.
While all denitrifying bacteria contain one or more types of Nor [14, 139], non-denitrifiers can also carry a Nor, qNor [98, 140]. qNor is a virulence factor that is needed for detoxification of ambient NO [140-142]. Previous work [97] showed that CuANor also confers resistance towards NO. The presence of CuA in the
enzyme [93] suggested a close relation to cytochrome oxidases. The recent determination of the sequence of its gene [139] indeed indicated that CuANor is
structurally related to cytochrome ba3 oxidase (Chapter 4, Fig. 1). In fact, CuANor
is much more closely related to cytochrome oxidases than any other Nor, although the enzyme lacks oxidase activity. Since cytochrome oxidases are electrogenic proton pumps we investigated the electrogenicity of CuANor from B.
azotoformans. In Chapter 4 it is shown that the enzyme indeed is electrogenic (Fig. 4). CuANor is the first Nor that was determined to be directly involved in the generation of a proton motive force. As a result, denitrifying bacilli produce more ATP per nitrate than Gram-negative bacteria.
16
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Chapter 2
Energy Transduction by Respiratory Metallo-enzymes: From Molecular Mechanism to Cell
Physiology
Sinan Al-Attar and Simon de Vries
Published: Coordination Chemistry Reviews, 2013, 257: 64-80
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ABSTRACT Membrane integrated respiratory metallo-enzymes occur in all three domains of life and perform the bioenergetic reactions sustaining the living state of the cell. To this end these enzymes catalyze redox chemical reactions that require tightly controlled electron transfer, proton binding and proton translocation events. The great quantity of structural and kinetic studies performed in the last two decades has provided the molecular detail needed for a basic understanding as to how respiratory enzymes convert redox free energy into a proton-motive force. In this review these recent developments are discussed within the framework of Peter Mitchell’s Chemiosmotic Hypothesis that is by now a Theory.25
Background
Physiology and bioenergetics
To sustain the living state, cells must supply themselves with energy sources from the surroundings such as nutrients or light. The central role of ATP in cellular metabolism as the universal energy donor, was established in the mid 1930s. However, and as first formulated by Peter Mitchell in 1961, the proton motive force (∆p or ∆µH+) plays a similar essential and universal role [1].
The cell’s metabolism can be divided into catabolic and anabolic chemical flows. Briefly, after transport into the cell, nutrients are broken down (catabolism) to yield both ATP and a limited set of biochemical building blocks (e.g Acetyl-CoA) that are subsequently assembled to a variety of biomacromolecules (anabolism). The free-energy flows of catabolism and anabolism are opposite and linked mechanistically and energetically through ATP and NADH. The majority of anabolic reactions employ NADPH rather than NADH and cells contain enzymes to convert NADH to NADPH thus enabling separate regulation of catabolic and anabolic flows.
In this simplified metabolic picture the cell is viewed as a scalar chemical free energy converter. However, the cell is a structure bounded by a semi-permeable membrane and, consequently, the uptake of nutrients and the excretion of metabolic end products are vectorial processes. The phospholipid portion of natural membranes is permeable to water, dissolved gases and certain small molecules in their uncharged state (e.g. acetic acid); in a metabolically active cell the concentration gradients of electrically neutral compounds across the membrane are generally close to zero. The uptake (or excretion) of all other compounds –charged or electrically neutral– requires the participation of specific proteins plugged through the cytoplasmic membrane, such as transporters for nutrients or minerals, respiratory chain enzymes for translocation of H+ or Na+
and secretory enzymes that translocate e.g. (partially) folded proteins.
The uptake of electrically neutral nutrients such as glycerol by transport proteins continues up to the point that the chemical potentials on either side of the membrane are the same (‘facilitated diffusion’). In case transport is mechanistically linked to the net input of chemical free energy in the form of e.g. ATP (‘active transport’), nutrients can be taken up against their gradient yielding a 100-1000 fold higher intracellular concentration. Charged nutrients equilibrate to electrochemical equilibrium and since the cell’s electrical capacitance is low, the chemical concentration gradient in equilibrium and even in steady state will remain low. However, any mechanism that compensates for the cell’s low
26
electrical capacitance can lead to ‘net transport’ of a charged compound to its physiologically relevant intracellular concentration. Transport mechanisms found in nature that compensate for the low electrical capacitance are antiport, i.e. exchange of compounds with the same charge, and symport, i.e. uptake of compounds with opposite charge; in both mechanisms the electrical component of the electrochemical gradient is annihilated. In addition, compensation of the low electrical capacitance may be accomplished in case two like charged compounds or a neutral and a charged compound with opposite chemical gradients are co-transported such as occurs in the symport of K+ and H+ or of
sugars and H+ [2]. Finally, transport may be supported by ‘brute force’ i.e.,
mechanistically linked to the hydrolysis of ATP.
The transport of nutrients underscores the free energy relations between chemical gradients, electrochemical gradients and the hydrolysis of ATP as well as the importance of their physiological equivalence. In the absence of ‘biochemical devices’ that enable free energy conversion, cellular metabolism, would be stalled. Primitive cells that would lack such devices had to be leaky in order to take up charged nutrients. The cell that we know today is the outcome of a series of evolutionary optimizations including an ion impermeable membrane and the development of thermodynamically efficient enzymes, that promote the rapid equilibration between (electro)chemical ion gradients and cellular ATP. This latter was recognized by Peter Mitchell in his Chemiosmotic Hypothesis proposing the existence of a reversible ATP synthase – nota bene as his postulate 1– driven by a proton electrochemical gradient (∆µH+) to produce ATP [1]. The
reversibility of the ATP synthase makes perfect physiological sense and indicates that both ATP and ∆µH+ should be regarded as ‘universal cellular energy donors’.
The evolutionary process has also produced organisms that use a sodium electrochemical gradient (∆µNa+) instead of ∆µH+ [3-6]; recently it has been
suggested that even both type of gradients might be employed in the same organism [7]. Bioinformatic analyses suggest that the sodium motive way of life may have predated the proton motive life style [8].
Redox metabolism
The sum reaction of all metabolic processes must be electroneutral. In general, nutrients are more reduced than biomass and to maintain the redox balance, excess reducing equivalents are excreted as partially reduced ‘waste products’ in addition to CO2. The amount of excess reducing equivalents related to the
anabolic formation of biomass constitutes a minor fraction (~ 10%) of the main flux of catabolic nutrient conversion linked to the generation of ATP. For example, growth on glucose in the absence of a terminal electron acceptor leads to the formation of the very oxidized CO2 and hence to a surplus production of
27
NADH. The latter is regenerated to NAD+ producing the typical main fermentation
products such as ethanol or lactic acid. The production of ATP in the fermentation of glucose proceeds via glycolytic substrate-level phosphorylation, a scalar reaction.
In the presence of terminal electron acceptors (e.g. O2, NO3-, fumarate) metabolic
electroneutrality is maintained mostly by the oxidation of (excess) NADH via respiratory chains. Furthermore, the oxidation of NADH leads to the production of ATP –much more than in fermentation– in the vectorial process called oxidative phosphorylation in which proton (or sodium) electrochemical gradients act as intermediate high-energy states. NADH is a water-soluble freely diffusible carrier of reducing equivalents (‘electrons’), in biochemical jargon often noted as ‘H-atom’ or ‘H2’. In the physiological sense, NADH is responsible for the cellular transport of electrons (though as the hydride anion); the term ‘electron transfer’ is used in the context of intra-enzyme electron transfer between fixed electron-donor and electron-acceptor pairs one of which may actually be bound NADH. Rates of electron transfer are well described by Marcus-like first order electron-tunneling expressions while rates of electron transport must, in addition, include second-order diffusion/collision terms [9-17]. Other freely diffusible water-soluble electron carriers in addition to NADH are low-potential ferredoxins, small c-type cytochromes, small blue-copper proteins and high-potential ferredoxins [18-20]. Likewise, the various ubi- and menaquinones, which are confined to the membrane through their isoprenoid chains, are membrane bound carriers of reducing equivalents. The latter freely diffusible membrane carriers are often erroneously referred to as ‘FADH2’ in textbooks.
The generation of excess reducing equivalents in metabolism has led to the concept of a central respiratory chain that funnels ‘electrons’ to the terminal electron acceptor [21]. The respiratory chain is basically a linear or branched cascade of soluble or membrane bound electron transfer enzymes. These enzymes are metabolically connected via the diffusible carriers that operate at different reduction potentials dictated by where precisely they interact with metabolism. Reduction potentials are approximately -0.4 – -0.3V for NADH/ferredoxin, -0.05 – 0.1 V for quinones, and 0.15 – 0.35 V for the small electron shuttles. Respiratory chains of prokaryotes (Bacteria and Archaea) are highly diverse because these organisms respond to fluctuating compositions and amounts of nutrients in the environment [18-20, 22]. In contrast, mammalian respiratory chains are invariably linear consisting of the ∆µH+ generating
enzymes Complex I or NADH dehydrogenase, the cytochrome bc1 complex and
the aa3-type cytochrome c oxidase electronically connected via ubiquinone and
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Krebs cycle or fatty acid metabolism, while cytochrome c exclusively shuttles electrons between the cytochrome bc1 complex and the cytochrome c oxidase, in
contrast to equivalent electron shuttles in prokaryotes that may serve several different reductases and oxidases. Mode of operation of direct and indirect chemiosmotic enzymes General background to the Chemiosmotic Theory
The aforementioned physiological perspective of the Chemiosmotic Theory, in particular the vectorial chemiosmotic coupling between producers and consumers of the proton motive force ∆µH+, has over a period of five decades
largely been experimentally verified and is now widely accepted. However, in respect to the precise biochemical mechanisms underlying ‘energy transduction’ and the quantitative aspects of the proton motive force ∆µH+, the views appear to diverge to this day (see also 2.5) and were first summarized in the multi-author Annual Review of Biochemistry issue of 1977 [23]. The term ‘chemiosmotic’ might be confusing but refers to reactions that are both chemical, such as transport/transfer of chemical groups (electrons, ‘H-atom’, oxo-, phosphate) and osmotic, for example the translocation of solutes or H+, rather than the movement of solvent across a membrane. The Chemiosmotic Hypothesis rested on four postulates summarized as [1, 2, 24, 25]: Reversible ATP synthases with a characteristic H+/ATP stoichiometry, respiratory enzymes with their
characteristic H+/e stoichiometry and solute transport systems each with a
characteristic H+/solute stoichiometry are embedded in a ‘coupling membrane’
(prokaryotic cytoplasmic membrane, mitochondrial or thylakoid (chloroplast) membrane) that is highly impermeable to ions, in particular to H+ or OH- (or Na+).
The direction of H+ translocation by respiratory enzymes is opposite to that for
phosphorylation of ADP to ATP by the FOF1-ATPsynthase. In order to achieve
‘directionality’ of proton translocation, the enzymes themselves must be oriented ‘asymmetrically’ in respect to the membrane, obtained for example by the location of substrate and product binding sites on opposite sides of the membrane. Though these latter structural features seem obvious today, they were not at the time of the proposal in 1961 when only the three-dimensional structure of Myoglobin was known and insight into the structure of membranes and membrane proteins was very rudimentary indeed.
Mitchell’s views about molecular mechanisms of energy transducing enzymes included physico-chemical concepts like group translocation, specific vectorial conduction, co-linear displacements of ligands, or in his own writing [24]:
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‘According to that unifying concept, vectorial metabolism is represented by a network of spatio-temporal pathways along which ligands (including solutes, ions, chemical groups, electrons, catalytic compounds and complexes) are conducted by articulated movements that occur in the direction of the thermodynamically natural escaping tendency, corresponding to the vectorial (or higher tensorial order) resultants of the thermodynamic and field-effect forces acting on the ligands’. In respect to mechanisms these concepts led to the proposal of the proton motive redox loop or the direct chemiosmotic mechanism to describe the action of respiratory enzymes. However, the direct mechanism turned out to be not correct (see below) to describe the action at the molecular level of the proton pumping ATP synthase, NADH dehydrogenase and cytochrome c oxidase.
The Proton motive Redox Loop
The basic features of the direct chemiosmotic mechanism are illustrated in the hypothetical scheme depicted in Fig. 1. The proton motive redox loop operates by the action of membrane diffusible electroneutral ‘H-atom’ redox couples A/AH2
and B/BH2 (for example Q/QH2 or O2/H2O) in conjunction with a transmembrane
electron transfer pathway between two redox centers located at opposite sides of the membrane such as two heme groups. The chemical and diffusion reactions that describe the direct chemiosmotic mechanism are given by: AH2(N) ⇔ AH2(P) (1) AH2(P) → 2H+P + 2e-(P) (2) 2e-(P) → 2e-(N) (3) B + 2e-(N) + 2H+N → BH2(N) (4) BH2(N) ⇔ BH2(P) (5) A(N) ⇔ A(P) (6) B(N) ⇔ B(P) (7) Sum: AH2 + B + 2H+N → A + BH2 + 2H+P (8) Here P and N refer to the positive (e.g. periplasm) and negative (e.g. cytoplasm) sides of the coupling membrane, respectively. Although the sum reaction (8) indicates a net movement of protons across the membrane, the reactions do not include a specific proton translocation or pumping step. If anything is being ‘pumped’, it is the electron that crosses the membrane. The net translocation of protons occurs as ‘H-atoms’ and is accomplished by the redox couples A/AH2 and
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Figure 1 Schematic drawing of the proton motive redox loop and the FO F1-ATP synthase representing
direct and indirect proton motive mechanisms, respectively. White arrows depict proton movements or proton translocation pathways (in the FOF1-ATP synthase) and red arrows show electron transfers. The
dotted white arrow indicates proton movement along the membrane. The N-side is the negative side of the membrane and the P-side the positive side. Protons at the P- or N-side or in the P or N bulk aqueous phases are designated as H+
P and H+N, respectively. The ATP synthase reaction is reversible indicated by
the double headed arrows and rotation of the shaft in two directions. Glu represents the conserved carboxylic acid residue of the c-subunit (there are 10 - 15 in total dependent on the organism), the proton entry and exit channels are located in the a-subunit.
within and across the membrane without energetic costs. It is imperative in this direct mechanism that electrons and protons ‘physically meet or diverge’ (reactions (4) and (2), respectively) at two different sides of the coupling membrane (N and P). Reactions (2) and (4) are basically scalar chemical reactions that occur in the P- and N-phases or at the membrane interface and are adequately described in terms of the midpoint potentials and pK-values of the participating chemical entities. In Mitchell’s view, the protons (reaction (2)) are ejected into the bulk aqueous P-phase, and do not migrate along the membrane (dotted arrow in Fig. 1). Examples of enzymes that have a transmembrane electron transfer pathway and operate according to the proton motive redox loop are the cytochrome bc1 and b6f complexes, the photochemical bacterial Reaction
Center, Photosystems I and II, nitrate reductase, formate dehydrogenase, Ni/Fe hydrogenases. Quinones universally serve as the H-atom carrier for these
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enzymes except for Photosystem I. The very movement of electrons across a low dielectric membrane, step (3), constitutes the energy transducing or energy conserving step. Step (3) is the only true vectorial step of the mechanism and itself leads to the formation of a transmembrane electrical potential (∆ψ). Ignoring reorientation of dipoles and changes in pK values as a result of the transmembrane electron transfer, the very binding of protons to ‘B2-‘ in step (4)
effectively moves net negative charge into the bulk aqueous N-phase, by removing positive charge (H+N), and thus conserves ∆ψ. Reaction (4) changes the chemical potential of protons in the bulk aqueous N-phase leading to a ∆pH. The total electrochemical difference, ∆µH+, between the two aqueous bulk phases N
and P or proton motive force (∆p) is thus given (in mV) by [1]: ∆p = ∆µH+/F = ∆ψ –2.3RT/F ·∆pH ~ ∆ψ – 60 · ∆pH at ~29°C (9) Since the cellular proton buffering capacity is much larger (~ 20 - 50 fold) than the electrical capacity the term ∆ψ (~ 200 - 250 mV) is much larger than ∆pH [2]. The thermodynamic driving force of a proton motive redox loop is given by the number of charges moving across the membrane (q) per electron transferred (q/e). The ratio q/e equals 2/2=1 for the example in Fig. 1, and in fact equals 1 for any proton motive redox loop because the pK values of e.g. Q/QH2 or O2/H2O differ substantially from the cellular pH~ 7. Sometimes the ratio /e (number of translocated or vectorial H+) is used instead of q, which is correct, but the ratio H+/e is not. The latter usually refers to the total number of protons appearing in the P-phase, where the pH is generally being measured, but this number includes the number of scalar protons liberated in reaction (2), which do not contribute to the overall useful thermodynamic work. The main reason for this is that the effective volume of the P-phase is much larger than that of the N-phase (bacterial cytoplasm, mitochondrial matrix or chloroplast lumen, ~ 1µm3 or less). The
P-phase may in reality be extending to the ‘Pacific Ocean’ because the outer membrane is permeable to protons and buffers and hence changes in electrochemical potentials are minimal. The small volume and low capacitance of the N-phase implies that the introduction or removal of charge produces a large transmembrane electrical potential that in practice is independent from the outside world. However, for protons moving along the membrane [26], the effective volume of the P-phase would be small. Protons that move along the membrane must therefore contribute to the total ∆p, for example as a surface potential, and hence perform thermodynamic work (see section 2.5 for a further discussion). H+
