Removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes, rebinding of CO dehydrogenase to depleted membranes, and restoration of respiratory activities

Abstract

In Pseudomonas carboxydovorans, CO dehydrogenase and hydrogenase were found in association with the cytoplasmic membrane in a weakly bound and a tightly bound pool. The pools could be experimentally distinguished on the basis of resistance to removal by washes in low-ionic-strength buffer. The tightly bound pool of the enzymes could be differentially solubilized under conditions leaving the electron transport system intact and with the nondenaturing zwitterionic detergent 3-(3-cholamidopropyl) dimethylammonio 1-propanesulfonic acid (CHAPS) and the nonionic detergent dodecyl ,B-D-maltoside. In vitro reconstitution of depleted membranes with the corresponding supernatants containing CO dehydrogenase led to binding of the enzyme and to reactivation of respiratory activities with CO. The reconstitution reaction required cations with effectiveness which increased with increasing ionic charge: monovalent (Li'), divalent (Mg2+, Mn2+), or trivalent (Cr3+, La3+). Reconstitution of depleted membranes with CO dehydrogenase was specific for CO-grown bacteria. Cytoplasmic membranes from H2-or heterotrophically grown Pseudomonas carboxydovorans had no affinity for CO dehydrogenase at all, indicating the absence of the physiological electron acceptor of the enzyme, which presumably is cytochrome b561, or another membrane anchor.Carbon monoxide dehydrogenase (CO dehydrogenase) is the key enzyme in CO metabolism of Pseudomonas carboxydovorans and other carboxydotrophic bacteria (for reviews, see references 7, 10-13, and 16). The enzyme catalyzes the oxidation of CO to CO2 (5) and provides two protons and two electrons (9,14,15) for the subsequent reduction of an electron acceptor which resides in the cytoplasmic membrane (13,17,18). Cytochrome b561 has been considered a strong candidate for the in vivo electron acceptor, as its redox potential is +40 mV (3, 4) and as it is involved in the CO-insensitive branch of the respiratory chain (for a review, see reference 12). After ultracentrifugation of crude extracts of P. carboxydovorans, more than 50% of CO dehydrogenase appeared in the soluble fraction (14,15). However, respiration rates with CO as electron donor could only be observed with membrane-bound CO dehydrogenase. Immunocytochemical localization employing exponential cells revealed 87% of CO dehydrogenase associated with the inner aspect of the cytoplasmic membrane (CM) and 13% located in the cytoplasm (17,18). When the bacteria entered the stationary growth phase or were disintegrated, more than 50% of the enzyme was released from the CM into the cytoplasm. Respiration rates were drastically reduced under such conditions (17). The basis for the association of CO dehydrogenase with the CM in vivo is so far unknown.A prerequisite for studying attachment and detachment of CO dehydrogenase to CMs of P. carboxydovorans in vitro was the availability of membranes devoid of CO dehydrogenase activity and of the protein itself. Such membranes should contain the complete and active electron transport system and retain the specific e...

The Mononuclear Molybdenum Enzymes

“…228 A portion of the CO 2 thus generated is subsequently fixed nonphotosynthetically via the reductive pentose phosphate pathway. 227 Aerobes such as O. carboxidovorans are responsible for a tremendous amount of bioremediation, accounting for the annual clearance of ~2 × 10 8 metric tons of CO from the environment. 229 The Mo-containing CO dehydrogenase from O. carboxidovorans and related organisms is distinct from the highly O 2 -sensitive Ni/Fe-containing CO dehydrogenase from obligate anaerobes such as Clostridum thermoaceticum or Methanosarcina barkerii.…”

Utilization of carbon monoxide by aerobes: recent advances

“…Depending on the growth phase, CO dehydrogenase in P. carboxydovorans can occur in the cytoplasm or attached to the inner aspect of the cytoplasmic membrane [7,22,23]. The enzyme was shown to associate with cytoplasmic membranes into two pools of different binding strength that are experimentally distinguished on the basis of resistance to removal by washes in low-ionicstrength buffer [12]. The tightly bound pool of the enzyme could be differentially soluhilized under conditio~ls leaving the electron transport system intact employing the nondenaturing zwitteronic detergent 3-[(3-cholamidopropyl)]dimethylammonio)l-propanesulfonate acid (CHAPS) and the nonionic detergent dodecyi ~-D-maltoside [24].…”

“…That CO dehydrogenase has hydrogenase activity was obvious from the ability of the electro-phoreticaUy homogeneous enzyme to catalyze the oxidation of H 2 [12,24]:…”

“…The basis of the process is not yet understood. However, we have recently achieved the in vitro removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes and rebinding of the enzyme to depleted membranes [12].…”

Principles of functional and structural organization in the bacterial cell: 'Compartments' and their enzymes

“…This change of enzyme location was paralleled by a drop of CO-oxidizing activity with 0 2. The CO-oxidizing activity with the unphysiological electron acceptor Methylene blue, which does not require contact of CO dehydrogenase with the membrane, always exceeded that with O 2 [49,52]. Measurements of respiration rates of extracts with different electron donors in addition to CO showed that the electron transport chain is not rate-limiting in intact cells, and the electron flow from CO to O~ is controlled by the amount of CO dehydrogenase attached to a membrane-bound physiological electron acceptor which finally turned out to be cytochrome b56 l (see below).…”