Camphor hydroxylation

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

P450CAM camphor-hydroxylating P450 from Pseudomonas putida... 

Gelb, M. H., Heimbrook, D. C., Malkonen, P., and Sligar, S. G. Stereochemistry and deuterium isotope effects in camphor hydroxylation by the cytochrome P450cam monoxygenase system. Biochemistry 21, 370-377 (1982). 

P450cam hydroxylates Ru-Cg-Ad when supplied with electrons via the natural NADH/putidaredoxin reductase/putd reduction relay.Ru-Cg-Ad hydroxylation occurs at only 1.6% the rate of camphor hydroxylation, and only 10% of the electrons supplied by NADH go to product formation. Presumably the rest are diverted to the formation of reduced oxygen species such as superoxide or hydrogen peroxide. The remarkable ability of P450cam to hydroxylate a molecule so structurally different from camphor supports the hypothesis that the structural flexibility inherent in the P450 fold allows these enzymes to hydroxylate structurally diverse substrates. 

Alton A, Shaik S, Thiel W (2007) What is the active species of cytochrome p450 during camphor hydroxylation QM/MM studies of different electronic states of compound I and of reduced and oxidized iron-oxo intermediates. J Am Chem Soc 129 8978... 

Oxygenation of the reduced cyt P-450(n)RH complex allows a second electron transfer from put which triggers camphor hydroxylation. 

Scheme 12.2 Fatty acid hydroxylation catalyzed by P450bm3 and camphor hydroxylation catalyzed by P450cam.

The catalytic efficiency of P450cam expressed in E. coli could be increased up to 25-fold by the coupling of its native electron transfer system (putidaredoxin reductase and putidaredoxin) to enzymatic NADH regeneration catalyzed by a recombinantly expressed glycerol dehydrogenase. This whole-cell system was applied for camphor hydroxylation in aqueous solution, as well as in a biphasic system with isooctane [181]. 

Sligar, S. G. (1975) A kinetic and equilibrium description of camphor hydroxylation by the P450cam monoxygenase system. Ph.D. Thesis, University of Illinois. 

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