Expression and Characterization of the Two Flavodoxin Proteins of Bacillus subtilis, YkuN and YkuP: Biophysical Properties and Interactions with Cytochrome P450 BioI
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- 作者：Rachel J. Lawson ; Claes von Wachenfeldt ; Ihtshamul Haq ; John Perkins ; and Andrew W. Munro
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：October 5, 2004
- 年：2004
- 卷：43
- 期：39
- 页码：12390 - 12409
- 全文大小：377K
- 年卷期：v.43,no.39(October 5, 2004)
- ISSN：1520-4995
文摘
The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressedin Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showedthat both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively)and that an error in the published B. subtilis genome sequence had resulted in an altered reading frameand misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue(neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometryconfirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reductionpotential for oxidized/semiquinone couple = -105 mV/-105 mV) with respect to the hydroquinone(midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV). Apoflavodoxinforms were generated by trichloroacetic acid treatment. Circular dichroism studies indicated that flavinmononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN.Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins.Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderatedestabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuPapoflavodoxin has similar (or slightly higher) stability compared to the holoprotein. Differential scanningcalorimetry reveals further differences in structural stability. YkuP has a lower melting temperature thanYkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic. Optical andfluorimetric titrations with oxidized flavodoxins revealed strong affinity (Kd values consistently <5 
M)for their potential redox partner P450 BioI, YkuN showing tighter binding. Stopped-flow reduction studiesindicated that the maximal electron-transfer rate (kred) to fatty acid-bound P450 BioI occurs from YkuNand YkuP at ~2.5 s-1, considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuNor YkuP, fatty acid-bound P450 BioI, and E. coli NADPH-flavodoxin reductase indicated that bothflavodoxins supported lipid hydroxylation by P450 BioI with turnover rates of up to ~100 min-1 withlauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supportedmonohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary productwas observed with both palmitic acid and palmitoleic acid as substrates.
M)for their potential redox partner P450 BioI, YkuN showing tighter binding. Stopped-flow reduction studiesindicated that the maximal electron-transfer rate (kred) to fatty acid-bound P450 BioI occurs from YkuNand YkuP at ~2.5 s-1, considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuNor YkuP, fatty acid-bound P450 BioI, and E. coli NADPH-flavodoxin reductase indicated that bothflavodoxins supported lipid hydroxylation by P450 BioI with turnover rates of up to ~100 min-1 withlauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supportedmonohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary productwas observed with both palmitic acid and palmitoleic acid as substrates.