The isolation and purification of cytochrome ~5 5 0 from the methylamine-oxidizing electron-transport chain in Thiobacillus versutus is reported. The cytochrome is a single-heme-containing type I cytochrome c with a relative molecular mass of 16 f 1 kDa, an isoelectric point of 4.6 f 0.1, a midpoint potential of 272 3 mV at pH < 4 and 255 f 5 mV at pH = 7.0, and an axial coordination of the Fe by a methionine and a histidine. The midpoint potential decreases with increasing pH due to the deprotonation of a group tentatively identified as a propionate (pK, = 6.5 f 0.1 and 6.7 f 0.1 in the oxidized and reduced protein, respectively) and a change in the Fe coordination at pH > 10. The electron-self-exchange rate appears to depend strongly on the ionic strength of the solution and is relatively insensitive to changes in pH. At 313 K and pH 5.2 the electron-exchange rate amounts to 0 . 7~ lo2 M-' s-' and 5.3 x lo2 M -l s-' at Z = 40 mM and Z = 200 mM, respectively. Amino acid composition and molar absorption coefficients at various wavelengths are reported. Resonances of heme protons and the & H 3 group of the ligand methionine of the Fe have been identified in the 'H-NMR spectrum of the reduced as well as the oxidized cytochrome.Although the composition and mode of operation of the methylamine-oxidizing electron-transport chain of Thiobacillus versutus is at present subject of intensive research, it is clear that the chain consists of only a few proteins and constitutes an attractive model system for studying biological electron transfer in vitro [l]. To this latter purpose the various members of the redox chain must first be identified and characterized. This paper deals with the isolation, purification and characterization of one of the proteins of this chain.The methylamine-oxidizing electron-transport chain in T. versutus is made by the bacterium when it is offered methylamine as its sole source of energy, nitrogen and carbon. The first enzyme in the chain is a methylamine dehydrogenase (MADH), a 123.5-kDa large quinone-containing enzyme with an a2Pz subunit structure, consisting of two identical monomers (up) which converts the amine to the corresponding aldehyde [2]. It has been isolated and characterized by Duine and coworkers [2], and its three-dimensional structure is under investigation [2, 31.The electrons liberated in the first step of the reaction are eventually transferred to O2 as the final electron acceptor [l]. The terminal enzyme in the redox chain is a membrane-bound oxidase [l] and the pathway between this enzyme and MADH consists of least two electron-transfer proteins [4], one of which is a blue copper protein called amicyanin [5], and one of which is a cytochrome [6]. As will be shown here, the latter in the presence of amicyanin, and the latter protein has been assumed to play the role of electron carrier between MADH and the cytochrome [l]. Since amicyanin and the cytochrome are both relatively small proteins, they constitute a pair of physiological partners ideally suited for the study of ...

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Quinoprotein (2,7,9-tricarboxy-1H-pyrrolo-[2,3-f]quinoline-4,5-dione quinone form (PQQ)-containing) ethanol dehydrogenase (EDH) from Pseudomonas aeruginosa ATCC 17933 was purified to homogeneity. EDH has an alpha 2 beta 2 configuration and subunits comparable in size to those of methanol dehydrogenase (MDH). Compared with other PQQ-containing dehydrogenases, Ca2+ is rather loosely bound and it seems necessary for PQQ binding and stability of EDH. Two soluble cytochromes c were detected in extracts from ethanol-grown cells and both were purified. One of these has an alpha-band at 551 nm for its reduced form, the oxidized form being an excellent electron acceptor for the semiquinone form of EDH. Since this cytochrome is quite different from the already known cytochrome c551 (operating in nitrate respiration) of this organism, it is indicated here as cytochrome cEDH. Comparison of the N-terminal amino acid sequence of cytochrome cEDH with the complete sequence of cytochrome cL (the electron acceptor of MDH), cytochrome cH (the electron acceptor of cytochrome cL) and cytochrome c551 revealed some similarity only to internal stretches of amino acids of the last two. The other soluble cytochrome appeared to be the already-known cytochrome c556. Since it was not an electron acceptor for cytochrome cEDH (neither for EDH), cytochrome cH is lacking in the quinoprotein-EDH-ethanol oxidation system of P. aeruginosa. It seems, therefore, that the respiratory chains for MDH and EDH are different.

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