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Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam.

Nagano S, Shimada H, Tarumi A, Hishiki T, Kimata-Ariga Y, Egawa T, Suematsu M, Park SY, Adachi S, Shiro Y, Ishimura Y
Biochemistry 42 14507-14 (2003)
Related entries: 1iwi, 1iwj

Cited: 26 times
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Abstract

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

Reviews citing this publication (9)

  1. Heme enzyme structure and function. Poulos TL. Chem Rev 114 3919-3962 (2014)
  2. Structural biology of heme monooxygenases. Poulos TL. Biochem Biophys Res Commun 338 337-345 (2005)
  3. The role of the heme propionates in heme biochemistry. Guallar V, Olsen B. J Inorg Biochem 100 755-760 (2006)
  4. Structural biology of redox partner interactions in P450cam monooxygenase: a fresh look at an old system. Sevrioukova IF, Poulos TL. Arch Biochem Biophys 507 66-74 (2011)
  5. Spectroscopic studies of the cytochrome P450 reaction mechanisms. Mak PJ, Denisov IG. Biochim Biophys Acta Proteins Proteom 1866 178-204 (2018)
  6. Structural biology of p450-oxy complexes. Poulos TL. Drug Metab Rev 39 557-566 (2007)
  7. Rational and semi-rational engineering of cytochrome P450s for biotechnological applications. Xu LH, Du YL. Synth Syst Biotechnol 3 283-290 (2018)
  8. Fourier transform infrared spectroscopy as a tool to study structural properties of cytochromes P450 (CYPs). Jung C. Anal Bioanal Chem 392 1031-1058 (2008)
  9. Oxygen activation and redox partner binding in cytochromes P450. Poulos TL, Madrona Y. Biotechnol Appl Biochem 60 128-133 (2013)

Articles citing this publication (17)

  1. Structural basis for effector control and redox partner recognition in cytochrome P450. Tripathi S, Li H, Poulos TL. Science 340 1227-1230 (2013)
  2. CO, NO and O2 as Vibrational Probes of Heme Protein Interactions. Spiro TG, Soldatova AV, Balakrishnan G. Coord Chem Rev 257 511-527 (2013)
  3. Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis. Kuznetsov VY, Poulos TL, Sevrioukova IF. Biochemistry 45 11934-11944 (2006)
  4. Differential sensing of protein influences by NO and CO vibrations in heme adducts. Ibrahim M, Xu C, Spiro TG. J Am Chem Soc 128 16834-16845 (2006)
  5. L358P mutation on cytochrome P450cam simulates structural changes upon putidaredoxin binding: the structural changes trigger electron transfer to oxy-P450cam from electron donors. Tosha T, Yoshioka S, Ishimori K, Morishima I. J Biol Chem 279 42836-42843 (2004)
  6. Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation. Zhang W, Pochapsky SS, Pochapsky TC, Jain NU. J Mol Biol 384 349-363 (2008)
  7. Electrochemistry of cytochrome P450 enzyme on nanoparticle-containing membrane-coated electrode and its applications for drug sensing. Liu S, Peng L, Yang X, Wu Y, He L. Anal Biochem 375 209-216 (2008)
  8. Crystal structures and functional characterization of wild-type CYP101D1 and its active site mutants. Batabyal D, Poulos TL. Biochemistry 52 8898-8906 (2013)
  9. Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy. Tosha T, Kagawa N, Arase M, Waterman MR, Kitagawa T. J Biol Chem 283 3708-3717 (2008)
  10. Investigation of the low frequency dynamics of heme proteins: native and mutant cytochrome P450(cam) and redox partner complexes. Karunakaran V, Denisov I, Sligar SG, Champion PM. J Phys Chem B 115 5665-5677 (2011)
  11. Molecular dynamics of the P450cam-Pdx complex reveals complex stability and novel interface contacts. Hollingsworth SA, Poulos TL. Protein Sci 24 49-57 (2015)
  12. Molecular recognition of ketamine by a subset of olfactory G protein-coupled receptors. Ho J, Perez-Aguilar JM, Gao L, Saven JG, Matsunami H, Eckenhoff RG. Sci Signal 8 ra33 (2015)
  13. A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s. Batabyal D, Lewis-Ballester A, Yeh SR, Poulos TL. Biochemistry 55 6517-6523 (2016)
  14. An Intermediate Conformational State of Cytochrome P450cam-CN in Complex with Putidaredoxin. Chuo SW, Wang LP, Britt RD, Goodin DB. Biochemistry 58 2353-2361 (2019)
  15. Conformational Change Induced by Putidaredoxin Binding to Ferrous CO-ligated Cytochrome P450cam Characterized by 2D IR Spectroscopy. Ramos S, Basom EJ, Thielges MC. Front Mol Biosci 5 94 (2018)
  16. Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam. Basom EJ, Manifold BA, Thielges MC. Biochemistry 56 3248-3256 (2017)
  17. Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin. Mammoser CC, Ramos S, Thielges MC. Biochemistry 60 1699-1707 (2021)


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