The Role of Water Molecules in the Association of Cytochrome P450cam with Putidaredoxin

Abstract

We have investigated the osmotic pressure dependence of the association between ferric cytochrome P450cam and putidaredoxin (Pdx) to gain an insight into the role of water molecules in the P450cam-reduced Pdx complexation amenable to physiological electron transfer. The association constant was evaluated from the electron transfer rates from reduced Pdx to P450cam. The natural logarithm of the association constant K a was linearly reduced by the osmotic pressure, and osmotic stress yields uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the association in the nonphysiological redox partners P450cam and oxidized Pdx. Although general proteinprotein associations proceed through dehydration around the complex interface, the interfacial waters could mediate hydrogen-bonding interactions. Therefore, about 10 more interfacial waters imply an additional water-mediated hydrogen-bonding network in the P450cam⅐reduced Pdx complex, which does not exist in the complex with oxidized Pdx. It is also possible that the water-mediated hydrogen-bonding interactions support a high P450cam affinity for reduced (K a ‫؍‬ 0.83 M ؊1 ) relative to oxidized (K a ‫؍‬ 0.058 M ؊1 ) Pdx. This study points to a novel role of solvents in assisting redox state-dependent interaction between P450cam and Pdx.

Putidaredoxin-to-Cytochrome P450cam Electron Transfer: Differences between the Two Reductive Steps Required for Catalysis

“…Importantly, in contrast to earlier studies (9,57-59), our results and data reported recently by others show that ET2 is considerably faster than ET1 at any Pdx: P450cam ratio (9,10,40,54,(60)(61)(62)(63) NIH-PA Author Manuscript NIH-PA Author Manuscript…”

“…The second dramatic difference between the two reductive steps was that the D38A, W106A, and Δ106 mutations led to >99% drop in ET2 k cat . Taken together, the kinetic results indicate that (i) Asp38 and Trp106 are much more important in ET2 than in ET1 and (ii) that the P450cam-Pdx r complexes formed during the two reductive steps must be structurally different and/or use different ET paths.Importantly, in contrast to earlier studies (9,57-59), our results and data reported recently by others show that ET2 is considerably faster than ET1 at any Pdx: P450cam ratio (9,10,40,54,(60)(61)(62)(63) NIH-PA Author Manuscript NIH-PA Author Manuscript …”

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…Several demonstrations of g L scale titers by engineered pathways show that, with careful optimization, cyanobacteria are promising hosts for heterologous NADPH-dependent metabolite production. These examples include the production of ethanol using alcohol dehydrogenase (Gao et al 2012), stereoselective reduction of various alkenes by an enoate reductase (Köninger (Kastritis et al 2011), 6 of which are electron transfer complexes, publically available from the Protein-Protein Interaction Affinity Database 2.0 website (http://bmm.crick.ac.uk/~bmmadmin/ Affinity/), and 10 additional electron transfer complexes (Holden et al 1994;Furukawa and Morishima 2001;Sétif 2006;Goñi et al 2009;Farooq and Roberts 2010). Gray crosses show non-redox complexes, and colored circles depict the 16 electron transfer complexes.…”

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

“…On the other hand, the addition of rat liver cytochrome b 5 (cyt b 5 ) and bacterial rubredoxins to oxy-P450cam can yield the hydroxylation product, whereas these non-physiological electron donors cannot transfer the first electron to ferric P450cam. Thus, the specific complex formation between P450cam and Pdx is required for the turnover reaction.We have studied the mechanism of the specific complex formation between P450cam and Pdx (19,20) and clarified that the P450cam⅐Pdx system exhibits unique features compared with other protein⅐protein ET complexes. Aoki et al (19) have reported the negative values for the changes in the enthalpy and entropy for the formation of the P450cam⅐Pdx complex determined from the isothermal titration and suggested that…”

“…We have studied the mechanism of the specific complex formation between P450cam and Pdx (19,20) and clarified that the P450cam⅐Pdx system exhibits unique features compared with other protein⅐protein ET complexes. Aoki et al (19) have reported the negative values for the changes in the enthalpy and entropy for the formation of the P450cam⅐Pdx complex determined from the isothermal titration and suggested that…”