Observation of the O-O stretching Raman band for cytochrome P-450cam under catalytic conditions.
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Dioxygen stretching (voo) Raman band was observed for the oxy form of Pseudomonas putida cytochrome P-450 (P-450cam) generated at room temperature under catalytic conditions, that is, in the presence of D-camphor, beta-NADH, putidaredoxin, and putidaredoxin reductase, by using the mixed flow transient Raman apparatus. At the same time the visible absorption spectra were monitored for the transient species. It was found that the voo frequency is little altered by binding of putidaredoxin to P-450cam, although the reduction rate of the oxy form becomes faster. Another intermediate with an oxygen isotope-sensitive band was not found in a time region until 2 s after mixing of the reduced enzyme with oxygen. (+info)
Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam. Indications for a similar overall topology.
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An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam). All sequences available on-line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography. While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms. Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms. Other helices which seem to exist in all P-450 forms include helices C, D, G and J. K. In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences. (+info)
Molecular characterization of a class I P450 electron transfer system from Novosphingobium aromaticivorans DSM12444.
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Elucidating the role of the proximal cysteine hydrogen-bonding network in ferric cytochrome P450cam and corresponding mutants using magnetic circular dichroism spectroscopy.
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