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Recording and Simulating Proton-Related Metabolism in Bacterial Cell Suspensions.

ABSTRACT: Proton release and uptake induced by metabolic activities were measured in non-buffered cell suspensions by means of a pH electrode. Recorded data were used for simulating substrate turnover rates by means of a new freeware app (proton.exe). The program applies Michaelis-Menten or first-order kinetics to the metabolic processes and allows for parametrization of simultaneously ongoing processes. The simulation includes changes of the transmembrane ΔpH, membrane potential and ATP gains, and demonstrates the principles of chemiosmotic energy conservation. In our experiments, the versatile sulfate-reducing bacterium Desulfovibrio desulfuricans CSN (DSM 9104) was used as model organism. We analysed sulfate uptake by proton-sulfate symport, scalar alkalinization by sulfate reduction to sulfide, as well as nitrate and nitrite reduction to ammonia, and electron transport-coupled proton translocation. Two types of experiments were performed: In oxidant pulse experiments, cells were kept under H₂, and micromolar amounts of sulfate, nitrate or nitrite were added. For reductant pulse experiments, small amounts of H₂-saturated KCl were added to cells incubated under N₂ with an excess of one of the above-mentioned electron acceptors. To study electron-transport driven proton translocation, the membrane potential was neutralized by addition of KSCN (100 mM). H⁺/e^- ratios of electron-transport driven proton translocation were calculated by simulation with proton.exe. This method gave lower but more realistic values than logarithmic extrapolation. We could verify the kinetic simulation parameters found with proton.exe using series of increasing additions of the reactants. Our approach allows for studying a broad variety of proton-related metabolic activities at micromolar concentrations and time scales of seconds to minutes.

SUBMITTER: Cypionka H

PROVIDER: S-EPMC8117226 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Proton transport (PTR) processes play a major role in bioenergetics and thus it is important to gain a molecular understanding of these processes. At present the detailed description of PTR in proteins is somewhat unclear and it is important to examine different models by using well-defined experimental systems. One of the best benchmarks is provided by carbonic anhydrase III (CA III), because this is one of the few systems where we have a clear molecular knowledge of the rate constant of the PTR process and its variation upon mutations. Furthermore, this system transfers a proton between several water molecules, thus making it highly relevant to a careful examination of the "proton wire" concept. Obtaining a correlation between the structure of this protein and the rate of the PTR process should help to discriminate between alternative models and to give useful clues about PTR processes in other systems. Obviously, obtaining such a correlation requires a correct representation of the "chemistry" of PTR between different donors and acceptors, as well as the ability to evaluate the free energy barriers of charge transfer in proteins, and to simulate long-time kinetic processes. The microscopic empirical valence bond (Warshel, A., and R. M. Weiss. 1980. J. Am. Chem. Soc. 102:6218-6226; and Aqvist, J., and A. Warshel. 1993. Chem. Rev. 93:2523-2544) provides a powerful way for representing the chemistry and evaluating the free energy barriers, but it cannot be used with the currently available computer times in direct simulation of PTR with significant activation barriers. Alternatively, one can reduce the empirical valence bond (EVB) to the modified Marcus' relationship and use semimacroscopic electrostatic calculations plus a master equation to determine the PTR kinetics (Sham, Y., I. Muegge, and A. Warshel. 1999. Proteins. 36:484-500). However, such an approximation does not provide a rigorous multisite kinetic treatment. Here we combine the useful ingredients of both approaches and develop a simplified EVB effective potential that treats explicitly the chain of donors and acceptors while considering implicitly the rest of the protein/solvent system. This approach can be used in Langevin dynamics simulations of long-time PTR processes. The validity of our new simplified approach is demonstrated first by comparing its Langevin dynamics results for a PTR along a chain of water molecules in water to the corresponding molecular dynamics simulations of the fully microscopic EVB model. This study examines dynamics of both models in cases of low activation barriers and the dependence of the rate on the energetics for cases with moderate barriers. The study of the dependence on the activation barrier is next extended to the range of higher barriers, demonstrating a clear correlation between the barrier height and the rate constant. The simplified EVB model is then examined in studies of the PTR in carbonic anhydrase III, where it reproduces the relevant experimental results without the use of any parameter that is specifically adjusted to fit the energetics or dynamics of the reaction in the protein. We also validate the conclusions obtained previously from the EVB-based modified Marcus' relationship. It is concluded that this approach and the EVB-based model provide a reliable, effective, and general tool for studies of PTR in proteins. Finally in view of the behavior of the simulated result, in both water and the CA III, we conclude that the rate of PTR in proteins is determined by the electrostatic energy of the transferred proton as long as this energy is higher than a few kcal/mol.

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Recording and Simulating Proton-Related Metabolism in Bacterial Cell Suspensions.

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