Ex. 14 Oxidase Test Objectives: ?? Cytochrome oxidases help with transfer of electrons from cytochromes to oxygen.

Electron Transport Phosphorylation Proton gradients store energy, both because of charge separation and concentration differential, and protons would rapidly cross the membrane to restore equilibrium if allowed. The cell membrane is impermeable to protons, except through protein complexes called ATP synthases. When protons move through these complexes, energy released by their passage is coupled to synthesis of ATP from ADP and phosphate (Pi). The exact details of this coupling are not yet clear, although it is known that part of the ATP synthase complex rotates during transport. The animation is not intended to be an accurate scale model of the details of the process, only to sketch the major concept, that entry of protons provides the energy needed to make ATP. This process is often called oxidative phosphorylation, since oxygen is a frequent electron acceptor, but is more accurately called chemiosmotic phosphorylation, since phosphorylation is coupled to the discharge of a chemiosmotic gradient. Animation 3 shows: Step 2: Protons (indicated by + charge) enter back into the bacterial cytoplasm through channels in ATP synthase enzyme complex. This entry is coupled to ATP synthesis from ADP and phosphate (Pi) Key notes for Animations 2 and 3: Protons are first translocated across the membrane, from the cytoplasm to the periplasmic space, as a result of electron transport resulting from the formation of NADH by oxidation reactions. (See animation of electron transport (Animation 1) if you don't understand this step.) The continued buildup of these protons creates a proton gradient. ATP synthase is a large protein complex with a proton channel that allows re-entry of protons. ATP synthesis is driven by the resulting current of protons flowing through the membrane: ADP + Pi ---> ATP Textbook Fig 5.16

Oxidase Test Determines presence of certain cytochrome oxidases Use whenever Gram-neg. bacteria have to be identified When test reagent gets oxidized it changes color. Oxidase test is positive if color change within 15 seconds!

Table 15-1: Oxidase Test Results and Interpretation Symbol Dark blue within 15 seconds Cytochrome c oxidase is present + No color change to blue or blue after 15 seconds Cytochrome c oxidase is absent ­ Oxistrip Only use on young cultures

The End Materials needed per table: Oxidase reagent (bottle with dropper) Small pieces of paper towel sterile Q-tips (2 per student) Pure slant culture needed per table: E. coli Pseudomonas aeruginosa Each student should individually do the oxidase test for both bacterial species. The End