Increasing P450Redox Partner Complexity Flavodoxins and Diverse Ferredoxins

The requirement for a single electron donor system for the P450s is met by fer-redoxin enzymes of the 2Fe-2S class in the mitochondrial adrenodoxin system, and in the well-characterized bacterial P450cam/putidaredoxin system. However, ferredoxins with other types of iron-sulfur clusters are found in nature, and the 3Fe-4S and 4Fe-4S ferredoxins are widespread in different organisms. Ferredoxins with these clusters have been shown to support P450 systems. For instance, the 4Fe-4S ferredoxin Fer was shown to drive fatty acid oxygenation catalyzed by Bacillus subtilis P450 Biol (CYP107H1) [99, 100]. Also, 3Fe-4S ferredoxins are known to support catalysis in, for example, the Mycobacterium tuberculosis CYP51 (sterol demethylase) P450 [101, 102]. In the M. tuberculosis system the gene encoding the 3Fe-4S ferredoxin (Fdx) is next to the P450 on the genome (genes Rv0763c and Rv0764c, respectively) and is cotranscribed.

Ferredoxins and flavodoxins have similar (and frequently overlapping) functions in prokaryotes, for example in nitrogen fixation reactions (e.g. 103). Flavo-doxins have capacity to accept either one or two electrons, forming semiquinone (SQ) or hydroquinone (HQ) states, respectively. However, they usually act as single electron carriers in biological systems, often shuttling between HQ/SQ forms [104]. In many cases, the redox potentials of the flavodoxins' HQ/SQ couples are also compatible with reduction of P450 heme [105]. Thus, it was not unexpected that flavodoxins might support catalysis of certain bacterial P450s, particularly in view of the evolutionary and structural relationships between fla-vodoxins and the FMN-binding (P450 electron donor) domains of eukaryotic and prokaryotic CPR modules [46]. In the B. subtilis Biol system, both host flavodoxins (YkuN and YkuP) support fatty acid oxygenation by the P450 (CYP107H1) [15, 106]. The flavodoxin cindoxin was also demonstrated to be the natural redox partner for P450cin in Citrobacter braakii, and its gene is located chromosomally adjacent to that for its partner enzyme. P450cin catalyzes cineole oxidation in a pathway enabling Citrobacter braakii to use cineole as an energy source for growth [107]. Pivotal studies in the Waterman laboratory also showed that mammalian

P450s expressed in E. coli had their oxygenase activities supported by a system comprising the bacterium's NADPH-flavodoxin reductase and flavodoxin proteins [e.g. 108].

The adaptability and evolvability of the prokaryotic P450s is manifest in their exploitation of a diverse range of cellular electron donor proteins. Key factors are clearly the evolution of a productive interaction site involving the proximal face of the P450, and the ability of the relevant flavodoxin/ferredoxin to donate single electrons to the heme iron at appropriate potentials.

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