Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni.
Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)
de Jong
G A
GA
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Caldeira
J
J
Sun
J
J
Jongejan
J A
JA
de Vries
S
S
Loehr
T M
TM
Moura
I
I
Moura
J J
JJ
Duine
J A
JA
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006418:Heme / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D045542:PQQ Cofactor; D011485:Protein Binding; D011487:Protein Conformation; D015363:Quinolones / Q000378:metabolism*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D001051:Apoenzymes; D003067:Coenzymes; D015363:Quinolones; C031690:heme C; D006418:Heme; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)