A scanning tunneling microscopy (STM) investigation of complex formation between cytochrome P450(cam) and putidaredoxin.
We have previously reported the scanning tunnelling microscopy (STM) imaging under buffer of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida [Faraday Discuss. 116 (2000) 1]. We describe here the adsorption and STM imaging under buffer of complexes of a mutant of cytochrome P450(cam), K344C, and wild-type putidaredoxin (Pdx) on gold(111). The images of Pdx on its own on gold(111) are not uniform, presumably due to multiple orientations of protein adsorption because of the presence of five or more cysteines on the protein surface. STM imaging of a 1:1 mixture of P450(cam)-K344C/Pdx showed a regular array of pairs of different-sized proteins 20-25 A apart arranged in rows across the gold(111) surface which we attribute to the P450(cam)/Pdx complex. The images of the pairs are more regular than those of Pdx on its own, probably as a result of complex formation with P450(cam) partly overcoming the heterogeneity of Pdx adsorption. As far as we are aware this is the first report of STM imaging of a protein/protein complex, and the first direct observation of P450(cam)/Pdx complex formation which is a key step in the catalytic cycle of P450(cam) catalysis. The redox centers of the two proteins are ca. 20 A apart, too far for rapid intracomplex electron transfer. Whether the observed complex is competent for electron transfer or physiologically relevant is not known, and further work is in progress to elucidate the protein-protein interaction.
Djuricic
Dejana
D
Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QH, UK.
Hill
H Allen O
HA
Lo
Kenneth K-W
KK
Wong
Luet Lok
LL
D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D006046:Gold; D016252:Microscopy, Scanning Tunneling; D009154:Mutation; D016958:Pseudomonas putida / Q000201:enzymology*; D013499:Surface Properties
D005288:Ferredoxins; C100137:putidaredoxin; D006046:Gold; D019475:Camphor 5-Monooxygenase