Substrates modulate the rate‐determining step for CO binding in cytochrome P450cam (CYP101)

Abstract

The high-pressure stopped-flow technique is applied to study the CO binding in cytochrome P450cam (P450cam) bound with homologous substrates (1R-camphor, camphane, norcamphor and norbornane) and in the substrate-free protein.The activation volume DV # of the CO on-rate is positive for P450cam bound with substrates that do not contain methyl groups. The k on rate constant for these substrate complexes is in the order of 3 · 10 6 M )1 AEs )1 . In contrast, P450cam complexed with substrates carrying methyl groups show a negative activation volume and a low k on rate constant of % 3 · 10 4 M )1 AEs )1 . By relating k on and DV # with values for the compressibility and the influx rate of water for the heme pocket of the substrate complexes it is concluded that the positive activation volume is indicative for a loosely bound substrate that guarantees a high solvent accessibility for the heme pocket and a very compressible active site. In addition, subconformers have been found for the substrate-free and camphane-bound protein which show different CO binding kinetics.Keywords: high-pressure stopped-flow; cytochrome P450; CO ligand binding; protein dynamics.Cytochromes P450 represent a big superfamily of heme-type monooxygenases that catalyze the conversion of diverse substrates [1]. Besides the main route of the reaction cycle from the substrate to the product there are side reactions which lead to the production of cytotoxic oxygen species such as hydrogen peroxide or of water in the oxidase reaction. These so-called uncoupling processes have been observed in many cytochrome P450 systems [2]. However, the structural parameters of the protein and the substrate which are responsible for the uncoupling process are not well understood. Data are increasingly accumulated indicating that the dynamics of the protein structure and in particular the accessibility of the active site for water molecules are very important [3]. In the oxidized form of P450 the high-spin/low-spin state equilibrium reflects a time-averaged population of water molecules at the sixth iron co-ordination site. This equilibrium can be monitored using the heme Soret band [4]. However, for the ironreduced form there is no spectral signal that could be used directly to monitor the water exchange. An indirect method is a water replacement technique using a probe molecule. In a large number of studies [4-9] using different approaches we found that the CO iron ligand is a good probe for the polarity and therefore for the presence of water molecules in the heme environment of cytochrome P450cam.To get a further insight into the dynamics of the water exchange process in different substrate P450 complexes we used the high-pressure stopped-flow technique [10,11]. The activation volume as well as the rate constant for the CO on-reaction obtained from such studies should allow us to quantitate dynamic properties of the heme pocket when P450 complexed with homologous substrates is studied. High-pressure flash photolysis studies on ferrous heme model complexes and hem...

Formation of Transient Oxygen Complexes of Cytochrome P450 BM3 and Nitric Oxide Synthase under High Pressure

“…In this article, stopped-flow kinetics of oxygen-binding under high pressure are reported for the first time. In the past, we had used high pressure stopped-flow (HPSF) to study the mechanism of CO-binding to heme-thiolate enzymes (Lange et al, 1994;Jung et al, 2002). The work with oxygen became feasible now after an instrumental adaptation (use of a ruby sphere acting as an inner valve), and by a very careful design of anaerobic experimental conditions: the concentration of dithionite must be high enough to ensure enzyme reduction, and sufficiently low to prevent reduction of oxygen.…”

“…Further support for the conformational explanation comes from flush-photolysis experiments of geminate CO rebinding to P450 and NOS, which gave evidence of doublet substate conformers of different CO-binding characteristics (Tétreau et al, 1997(Tétreau et al, , 1999. Moreover, a high pressure stopped-flow study of CObinding to P450cam also revealed the coexistence of conformers of different activation volumes DV z (Jung et al, 2002).…”

Reverse Spin‐Crossover and High‐Pressure Kinetics of the Heme Iron Center Relevant for the Operation of Heme Proteins under Deep‐Sea Conditions

“…Based on classical kinetics and thermodynamics we expect that the molecule binding to a vacant coordination site within the pockets of the enzymes is accelerated at high pressures and that a low‐spin state of heme proteins (usually in a form of the six‐coordinate aqua species) should be favored under such conditions 1b. 6 Both effects would have lethal consequences, because they would speed‐up respiration (metabolism in general) and promote undesired side‐reactions 2c. But biology shows us opposite.…”

“…availability) increases under elevated pressures because of the thermodynamic rules 10. This has a biological relevance because a controlled accessibility of the heme active sites in cytochrome P450s for water molecules is crucial in order to prevent undesired side processes which can generate cytotoxic reactive oxygen species 2c…”

Mechanistic Studies on the Activation of NO by Iron and Cobalt Complexes

“…Volume profiles for reversible NO binding to (a) the substrate-free form of P450 cam [44] and (b) complex 4 in methanol. [67] tein and the heme pocket to be more rigid and less compressible [57] ) and/or the much larger reorganization of spin multiplicity upon NO binding to substrate-bound P450 cam (from S = 5/2 to S = 0) than that observed for the substratefree analogue (from S = 1/2 to S = 0).…”