Modeling the electron transport chain of purple non-sulfur bacteria

Steffen Klamt¹, Hartmut Grammel¹, Ronny Straube¹, Robin Ghosh² & Ernst Dieter Gilles¹

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Synopsis

The attractiveness of anoxygenic non-sulfur photosynthetic bacteria (Rhodospirillaceae) for studying gene regulation in response to cellular redox events is based on their energetic flexibility, which enables growth under a variety of environmental conditions. In the presence of oxygen, electrons are transferred through a respiratory chain comprising NADH (or FADH), ubiquinone (Q), cytochrome bc₁, cytochrome c₂ and finally to oxygen by cytochrome oxidases or ubiquinol oxidases, thereby enabling phosphorylation of ADP via ATPase complexes. Note the functional homology to the mitochondrial respiratory chain. At low oxygen tensions, the biosynthesis of intracytoplasmic membranes (ICMs) is induced. Specific polypeptide-bacteriochlorophyll (BChl) components of these membranes––the photosynthetic light-harvesting complexes (LHCs) and reaction centers (RCs)––capture light energy, which drive a cyclic photophosphorylation system via RC-Q-cyt bc₁-cyt c₂ back to RC. It has been stated as early as 1957 that the redox state of components of the respiratory and photosynthetic electron transport chain (ETC) may constitute metabolic signals that govern the expression of LHC- and RC-encoding genes and therefore determine the adaptation of the cells to a given environmental condition (Cohen-Bazire et al, 1957). The repression of ICM synthesis under high oxygen tension and also anaerobically by high light intensities has been studied in detail during the last years and some regulatory systems involved were identified, including the two-component system RegA/RegB (PrrA/PrrB), the PpsR (CrtJ) repressor and the Fnr-like transcriptional regulator FnrL (Oh and Kaplan, 2000; Bauer et al, 2003). However, particularly for light regulation of ICM synthesis, the molecular signals and mechanisms involved in the initiation of signal transduction pathways governing photosynthetic gene expression are still poorly characterized or even unidentified. The high-light repression effect has been attributed to be dependent on the redox state of the membranous Q pool (Oh and Kaplan, 2000). However, to our knowledge, no direct experimental measurements of Q redox states under different light conditions are available so far. Recently, it was demonstrated that oxidized Q directly inhibits the RegB/RegA regulatory system of Rhodobacter capsulatus in vitro (Swem et al, 2006), thereby potentially mediating the repression of ICM synthesis also under high oxygen conditions in the dark.

The lack of quantitative information about how the redox states of individual ETC components change under different growth conditions limits the verification of the proposed hypotheses of redox regulation and a pure qualitative discussion of the behavior of the ETC is infeasible as too many variables are involved. Here, we present (i) a stoichiometric model of the ETC unambiguously describing the structural and functional capabilities of the ETC of purple non-sulfur bacteria and (ii) a kinetic model based on thermodynamic constraints of the underlying processes that enables us to simulate the steady-state behavior of the ETC, in particular the redox states of ETC components, under different environmental conditions.

Elementary-mode analysis (see Schuster et al, 2000) of the ETC reveals nine fundamental modes expressing all the potential functional behaviors of the ETC in the steady state. The stoichiometric model reproduces well-known cycles and pathways for the ETC when operating under photosynthetic (cyclic photosynthesis and reverse electron transport), respiratory (electron transfer from NADH or succinate to ubiquinol oxidase or cytochrome (cbb₃) oxidase) or fermentative (fumarate reduction) conditions. Apart from these well-known functions, the stoichiometric model revealed two operational modes representing reverse electron flow under respiratory conditions. So far, the functional role of reverse electron flow has been mainly discussed for photosynthetic growth only. In fact, due to thermodynamic constraints, reverse electron flow under aerobic conditions plays only a minor role and as shown by simulations of the kinetic model, the concentration ratios of the metabolites involved have to be at rather extreme levels, to make this state thermodynamically favorable. However, this operation might become important for certain (possibly temporary) metabolic states, for example, if a great imbalance in the current redox potentials of NADH and succinate occurs.

The stoichiometric model thus identified––in an unbiased way––the meaningful functions of the ETC; however, it can only show the potential behaviors of the ETC (which are under real conditions normally superimposed). For studying the actual driving forces, electron flows and redox states occurring in the ETC under different environmental conditions, we therefore constructed a kinetic model that also includes known regulatory pathways governing the expression of ETC components. Although in photosynthetic bacteria two types of energy-converting membranes (ICM and cytoplasmic membrane) are present, which are structurally and functionally different, we set up the model with only one membrane compartment. We justify this approach because we consider in the simulations only scenarios where the model functions either as a pure photosynthetic membrane (ICM model) (i.e. no participation of oxidases) or as a pure respiratory (cytoplasmic) membrane (no participation of photosynthetic RCs). The reaction steps taking place inside the enzyme complexes have been described with rather simple, yet thermodynamically correct kinetic laws based on the differences in the redox potentials of the participating redox couples. As boundary conditions for metabolites directly interacting with the ETC, we fix the concentration ratios of NADH/NAD, succinate/fumarate and ATP/ADP (which can, however, be varied for the different scenarios considered).

To our knowledge, it is the most comprehensive model of the ETC in purple bacteria with respect to the components and processes considered. Even though we cannot claim that the model is accurate when absolute quantitative units are considered (uncertainties in parameters and only few available measurements do not permit this), it provides valuable semiquantitative and qualitative insights into the behavior of the ETC, in particular on the redox states of key components which are often difficult to measure.

Important results of the steady-state analysis of the model are shown in Figure 5. The plots show the dependency of the redox states of the Q and cytochrome c₂ pools (Q_charge and c2_charge), of the proton-motive force (pmf) and BChl concentration (Bchl; as a measure of ICM) on the cytosolic redox state (represented by NADH/NAD) for four different growth conditions: aerobic and semi-aerobic in the dark and anaerobic under high-light and low-light conditions. The model is able to reproduce the observations about the ICM levels that are repressed during aerobic growth and are relieved from oxygen repression under semi-aerobic conditions. Importantly, the level of ICM corresponds to the reduction degree of Q. That oxygen-limiting conditions result in a more reduced Q pool compared to fully aerobic growth can be deduced in a straightforward manner from the operation of a linear respiratory chain. In contrast, the adaptation of the Q redox state when the cyclic photophosphorylation system faces different light intensities is not deducible simply by pure intuition. To our knowledge, the question whether a switch from high- to low-light conditions results in a more reduced (as the physiological regulatory model demands to explain high-light repression of ICM synthesis) or a more oxidized Q pool is currently not resolved by literature data. Strikingly, the simulation of this situation with the mathematical model yields an unambiguous result: the Q pool is reduced more under low-light conditions, which is in accordance to its proposed role as a metabolic signal for ICM expression (Swem et al, 2006). It is important to point out that this finding is virtually independent of the parameter values employed, instead it is an inherent feature of the system. We could not find a parameter set where the opposite result, that is Q gets reduced more under high-light conditions, is produced.

Figure 5

Simulated steady state response curves of selected model variables for a range of NADH/NAD ratios (R_NADH). Four different growth regimens were considered. Measurements are indicated by circles in the respective color.

Full figure and legend (167K)Figures & Tables index

Another conclusion that can be drawn from Figure 5 is, that (only) under photosynthetic conditions there is an optimal load of the ETC with electrons (and therefore an optimal cytosolic redox state here reflected by the ratio NADH/NAD) resulting in a maximum of p. The model clearly demonstrates that the optimal load is dependent on the light intensity. Such a biphasic behavior cannot be seen for respiratory growth: p increases monotonically with the cytosolic redox state.

These results illustrate the value and applicability of the model presented in deriving predictions on the behavior of the ETC in anoxygenic photosynthetic bacteria, which await experimental validation now.

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