The Roles of Putidaredoxin and P450cam in Methylene Hydroxylation

Putidaredoxin, the iron sulfur protein from the P450,,,mediated methylene hydroxylase system of Pseudomonas putida, is shown to be essential both as an electron transfer agent and as an effector of P450,,, necessary for product formation. P450,,, is recognized to progress through four well defined states in an ordered reaction cycle of oxidized, and, with substrate, oxidized, reduced, and oxygenated. Combination of reduced putidaredoxin with each of these states of P450,,,, or of oxidized putidaredoxin with the reduced or oxygenated P450,,, in the presence of substrate and 0 2 yields product stoichiometrically. Putidaredoxin is not replaced efficiently by other iron sulfur proteins, nor by the phospholipid from the hepatic microsomal P450 system. Rapid scan spectroscopy shows the oxygenated form of P450,m to be dominant during NADH turnover in the reconstituted system. A scheme is developed for the reaction cycle in which aggregates of the enzyme components are postulated to facilitate electron transfer and determine the nature of the product formed. Analytical treatment of steady state kinetic data suggests that a 1: 1: 1 equivalence of reductase to putidaredoxin to P450,,, is functional and probably reflects participation of 1 molecule each of putidaredoxin, P450,,,,, and possibly reductase in the catalytic complex.

to be dominant during NADH turnover in the reconstituted system. A scheme is developed for the reaction cycle in which aggregates of the enzyme components are postulated to facilitate electron transfer and determine the nature of the product formed.
Analytical treatment of steady state kinetic data suggests that a 1: 1: 1 equivalence of reductase to putidaredoxin to P450,,, is functional and probably reflects participation of 1 molecule each of putidaredoxin, P450,,,,, and possibly reductase in the catalytic complex.

Recent publications
have been concerned with the role of iron-sulfur proteins in multicomponent hydroxylase systems (l-3).
'These proteins have been inferred to function as intermediate transfer agents for electrons from a flavoprotein reductase to a specific terminal oxidase (4-10). The tendency to implicate iron-sulfur proteins as electron transfer agents derives mainly from their ability to stimulate turnover of reduced pyridine nucleotide in the presence of reductase (9, 11) in the reconstituted systems (4-10)) with reduction of either the terminal oxidase (11) or other acceptors, and also from their similar role in other elecron transfer processes. from steady state kinetic, stereochemical, and substrate specificity studies that only 1 electron for oxygen reduction is transferred by way of the iron-sulfur component, the 2nd being transferred directly to an oxygenated form of the terminal oxidase by reduced pyridine nucleotides (4,12). In drug and steroid hydroxylation in liver microsomes, cytochrome bs has been postulated to transfer the 2nd electron to an oxygenated P450 complex (13). In fatty acid hydroxylation by P450 in liver microsomes, an iron-sulfur protein is not utilized for electron transfer but evidence suggests that a phospholipid is required (14). Clarification of the role of iron-sulfur proteins is pivotal to the development of a model for biochemical action of multicomponent hydroxylase systems. This laboratory has been pursuing research directed toward understanding monoxygenase mechanisms by using the camphor hydroxylase system from Pseudomonas putida (10, 15).
Reports on our discovery of a specific requirement for putidaredosin in product formation and of the 418 nm oxygenated form of 1'450 have been given (16)lm3 and a reaction scheme for hydroxylation developed ( Fig. 1) (17).
The oxygenated form has also been reported independently elsewhere (18). One approach has been to dissect the reaction into the probableelements and to study the interactions of the separate elements. We have purified the three components of the monoxygenase system and have studied their interactions by chemical and kinetic techniques. The kinetics section of this work, including fast reaction and analytical kinetic methods applied to the reconstituted system, is reported in detail here.  pearance of the 432 nm band of 4-nitrocatechol in the presence of horseradish peroxidase and HzOs was alternately used.
No Hz02 has been detected. The D and L enantiomers of camphor, their 1,2-lactones, and several very similar bicyclic monoterpenes serve as substrates (24) 25" under air to an altered :uld catalytically inactive form of the ellz\-me called P420.
If the substrate is present, this con\-ersion does not occur rapidly.

Reduced Species
The 1'450,. 1,1 camphor complex can be reduced quantitatively with NAIDI-I in an anaerobic titration to an end point of 1 electron per heme with the formation of the 408 nm peak, t,,,M = 72 (15, 17). Concurrent with this reduction, the ferric iron epr signal disapllears (23). NhD+ concentration does not increase after the 1 electron end point.
1'450,,,, is also photoreducible to a species exhibiting the same spectral characteristics. The yelocities and reactants are shown in Table II.
In the full system without camphor present, NADII will reduce the reductase flavoprotein and the redoxin but not the cytochrome P450,,,,,. The 1'450.camphor complex, however, is readily reducible.
The maximal rate constants for reduction b) NADH of the components separately and in combination are given in Table III.
With reductase and redoxin both present, the rate of the cytochrome-camphor complex reduction is lo2 to lo3 times faster than by any other pathway and is nearly double the turnover jump experiment was designed to measure this rate in which NADH-reduced P450 Ca,n IS exposed to air and then jumped from 22" to 25" after 90 sec. At this time, 60% of the heme exists as oxygenated complex (see below).
The absorbance changes observed, which are in opposite directions at 390 and 420 nm, have associated relaxation times of 2 msec. This time shortens as the NADH and oxygenated complex disappear. A bimolecular rate constant of 1.7 X 1O+6 M-' see-' was calculated from the relaxation time.
Oxygenation is sufficiently fast for the 418 nm species to be an intermediate in the hydroxylase reaction but too fast for it to be the rate-determining step. In the absence of redoxin the oxidized P450,.>,,, camphor complex regenerates spontaneously from the oxygenated sljecies as shown in Fig. 2. This process is first order as determined by either Guggenheim (26)   In contrast, the oxygenated species disappears within milliseconds after the addition of an excess of reduced putidaredoxin (t+ < 10 msec). The decay rate with oxidized putidaredoxin is only slightly slower as reported by Peterson et al. (28). With either 30 pM Hz02 or cytochrome c, the kg is 0.02 se@, and with 300 PM HzOz, 0.06 see-i. The low reactivity of HzOz suggests a small rate constant for P450,,, deoxygenation, (L8), forotherwise HzOz, which reacts very fast with the reduced P450, would accelerate the conversion to the oxidized form. A k-3 of 0.01 see-I was calculated from the forward rate constant and a K D for 02 of about 5 mM, derived from the KD for CO, 50% inhibition with a CO-02 (1 :l) gas mixture (6) and the fact that CO is 40 times more soluble than 02 at 25".

Minimum
Requirements for Hydroxylation will reduce 1'450,,,n but fail to produce hydrosylnted product.

Role of putitlarecloxin in product formation
The conditions were as in Table II   At concentrations less than saturating for redoxin, in nearly equimolar concentration with reductase and P450,,,, the 418 nm oxygenated form is dominant as shown in Fig. 3.3 When NADH is depleted, the 418 nm band still dominates under the given conditions, thereafter changing back to the 391 nm form at a rate far slower than the turnover number for P450,,,.

Dependence of Rate on Ratio oj Enzyme
Components-The kinetic data (Figs. 4 and 5) and product yields given in Tables  IV to VI demonstrate  that both putidaredoxin  and P450,,,n are required for substrate hydroxylation.
The ratio of redoxin to the other components for hydroxylation is critical to optimize turnover.
Previous reports of the ratio of the enzyme components for maximum turnover under assay conditions with other reductase-redoxin-P450 hydroxylase systems were 1: 20 : 1 and 1:50: 1 (24, 29, 30). It is now found, however, that as the absolute concentrations of the components is increased, the relative amount of redoxin required to saturate the system decreases. Fig. 4 illustrates these results. The asymptotic value of redosin to reductase-P450 cam could not be obtained from these data, but the linearity of reciprocal plots and the 1: 1 break in saturation curves with reductase and P450,,, (not shown) suggest that the in vitro as well as in viva ratio of components could be 1:l:l. Simultaneous variation of the hydroxylase components in any ratio between 1:03 : 1 and at least 1:3 : 1 reductase to redoxin to P450,,, results in a nonlinear rate dependence as shown in Fig. 5. If one of the components in Fig. 5, such as the reductase, is held constant, then the rate dependence is linear on simultaneous variation of the other two components well past the 1 :l saturation point for the reductase and P450,,,. These results may be consistent either with an absolute increase in the amount of enzyme in the complexed form present at higher enzyme concentrations or possibly with a decrease in the mean free path between collisions needed to effect electron transfer.
Kinetic Evidence for Enzyme Complexes-The kinetic x-axis intercept of l/v versus l/[redoxin] plots at different, fixed reductase-P450,,, concentrations shown in Fig. 4 clearly suggests effector kinetics.
If it is assumed that some redoxin-P450 (1: 1) complex is vital to catalysis, KBc for this complex (the reciprocal of the intercept value) would be roughly 30 pM. One can take this value and use it to calculate constant turnover numbers from rate data at all concentrations of P450,, and nonsaturating redoxin levels rather than requiring the redoxin to be saturating, which supports the validity of the assumption of a 1:l complex between the two components. If, however, the ratio of reductase to P450,,, is increased, the x-axis intercept (although remaining constant for any fixed ratio) decreases ultimately to 4.2 pM, indicating that the intercept value contains other terms, the nature of which may be resolved in further study. 5. Rate dependence on enzyme concentration: simultaneous variation.
The conditions were as in Fig. 4 under similar conditions could be due to the formation of multiredoxin-hydroxylase complexes.
The linearity of the double reciprocal plots of NADH turnover versus redoxin concentration (Fig. 4), and the unique intercept on the abscissa for any given ratio of P450 to reductase suggest, however, that the active complex contains one putidaredoxin with a relatively high dissociation constant.
Rapid dissociation could facilitate the transfer of the 2 required electrons for hydroxylation by the strictly 1 electron-reduced putidaredoxin, although not necessarily by t,he same molecule.
The observation that quantitative yields of hydroxylated product are derived from combination of reduced putidaredoxin and either oxidized or oxygenated P450,,,ncamphor complex is the best experimental evidence to date that the electrons can be transferred in two steps. It is not clear whether reductase participates directly in the catalytic complex or transfers electrons to redoxin which in turn transfers them to P450, In via two sequential binary complexes. Studies are continuing to see whether a physical significance can be more clearly attached to the x-axis intercepts obtained in Fig. 4 over a much broader range of conditions and whether a general rate law for the over-all reaction can be developed.
Biophysical studies by Mossbauer, EPR, and ultracentrifuge studies are in progress to resolve the question of the capability of these components to complex and the detailed mode of electron transfer and oxygen activation.