Cytochrome partners

Nuclear Receptor Regulation of Hepatic Cytochrome P450 Enzymes. Figure 1 General mechanism for transcriptional activation of CYP genes by xenochemicals that activate their cognate xeno-receptor proteins. In the case of Ah receptor, the receptor s heterodimerization partner is Arnt, whereas in the case of the nuclear receptors CAR, PXR, and PPARa, the heterodimerization partner is RXR. The coactivator and basal transcription factor complexes shown are each comprised of a large number of protein components. 

Seeliger S, R Cord-Ruwisch B Schink (1998) A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as a ferric iron reductase and as an electron carried to other acceptors or to partner bacteria. J Bacteriol 180 3686-3691. 

In some cases, small biological redox partner proteins such as heme-containing cytochromes, ferredoxins comprising an iron-sulfur cluster, or azurin with a mononuclear Cu site have been used as natural mediators to facilitate fast electron exchange with enzymes. A specific surface site on the redox protein often complements a region on the enzyme surface, and enables selective docking with a short electron tunneling... 

The catalytic activity of CYP enzymes requires functional coupling with its redox partners, cytochrome P450 NADPH oxidoreductase (OR) and cytochrome bs. Measurable levels of these two proteins are natively expressed in most cell lines. Therefore, introduction of only the CYP cDNA is generally needed for detectable catalytic activity. However, the levels of expression of the redox partner proteins may not support maximal CYP catalytic activity, and therefore enhancement of OR levels may be desirable. This approach has been used successfully with an adenovirus expression system in LLC-PKi cells [12],... 

A redox reaction is a special case of the equilibrium reaction of A + B in Equation 13.1 B is now a reducible group in a biomolecule with an EPR spectrum either in its oxidized or in its reduced state (or both), and A is now an electron or a pair of electrons, that is, reducing equivalents provided by a natural redox partner (a reductive substrate, a coenzyme such as NADH, a protein partner such as cytochrome c), or by a chemical reductant (dithionite), or even by a solid electrode ... 

Other redox partners Co(bipy)33+ (oxidant) and Ru(NH3)s py2+ (reductant) are likewise partially blocked by Pt(NH3)6 +. Interestingly the reaction of cytochrome c(II) with PCu(II) is also blocked by Pt(NH3)5 +, thus identifying this as a site for electron transfer with cytochrome c. This observation is con-sis tant with a preliminary report of NMR results (19). The blocking is in fact more extensive than that observed with the above complexes, which is reasonable in view of the larger size of cytochrome c. Reaction with the negatively charged dipicol-inate oxidant, Co(dipic)2, was similarly investigated, where separate association of the oxidant with Pt(NH3)6 + can be... 

It is assumed in these experiments that the modification closest to the electron-transfer site will have most effect on rate constants. Rate constants are enhanced for 3+ and retarded for 3- redox partners. With Co(phen)33+ and Fe(CN)53 as oxidants it has been demonstrated that both react at the exposed heme edge of cytochrome c (23). The exposed heme edge is also relevant with PCu(II) as oxidant, Table III. With all three oxidants... 

D.R. McMillin, Purdue University In addition to the charge effects discussed by Professor Sykes, I would like to add that structural effects may help determine electron transfer reactions between biological partners. A case in point is the reaction between cytochrome C551 and azurin where, in order to explain the observed kinetics, reactive and unreactive forms of azurin have been proposed to exist in solution (JL). The two forms differ with respect to the state of protonation of histidine-35 and, it is supposed, with respect to conformation as well. In fact, the lH nmr spectra shown in the Figure provide direct evidence that the nickel(II) derivative of azurin does exist in two different conformations, which interconvert slowly on the nmr time-scale, depending on the state of protonation of the His35 residue (.2) As pointed out by Silvestrini et al., such effects could play a role in coordinating the flow of electrons and protons to the terminal acceptor in vivo. 

Scott, E.E., White, M.A., He, Y.A., Johnson, E.F., Stout, C.D. and Halpert, J. R. (2004) Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl) imidazole at 1.9-A resolution insight into the range of P450 conformations and the coordination of redox partner binding. The Journal... 

Cytochrome c and cytochrome c peroxidase (ccp) are physiological partners in the ccp reaction cycle structural, thermodynamic, and kinetic data are available for the protein-protein interaction [69-72]. A model indicates that the cyt c/ccp complex is stabilized by specific salt bridges with the hemes in parallel planes the Fe-Fe distance is 24 A, and the edge-edge distance is 16 A [70]. 

In all protein-protein complexes studied to date in which cytochrome c has been a partner, it has been shown that the ET rates depend strongly on the reaction driving force. It follows that variations in the reorganization energy could control ET rates in these cases [12]. In redox enzymes with two or more active centers, ET between two centers could be turned on by lowering X at roughly constant — AG [1]. Indeed, a proposal has been advanced that this type of mechanism would be an efficient way to gate the electron flow in a redox-linked proton pump such as cytochrome oxidase [75]. 

In protein-protein reactions, the donor-acceptor distance is determined by the structure of the reacting proteins, and the way(s) in which they bind and interact. For example, it is generally believed that cytochrome c binds to its reaction partners at or near the exposed heme edge, in order to minimize the reactant distance and thereby maximize the rate. The redox active centers of most proteins are sufficiently buried that the large protein imposed distances provide low intrinsic reactivity for the proteins with respect to exogenous... 

The His35, with coordinated His46 in close proximity, has frequently been suggested as a site for electron transfer reactivity of azurin. Two processes have been detected in a temperature-jump study on the equihbration of azurin with cytochrome C551, its physiological partner [57]. The fast process is assigned to electron transfer, and the slower process to a conversion between inactive and active forms of reduced azurin. It has been concluded that the active form is protonated. A second H-bonded form of His35 is believed to result from the protonation [2]. 

Whatever the explanation, the sensitivity of the remote pK to oxidation state of the Cu is of potential importance in relation to the functional role of plastocyanin. Plastocyanin and its physiological electron transport partner cytochrome f are believed to have complementary surfaces which lead to efficient interaction prior to electron transfer. As will be seen below there is substantial evidence for cytochrome f(II) (as reductant) reacting at the remote site of PCu(II). One problem which may be anticipated here is how dissociation of the product... 

The kinetics of the reactions of horse cytochrome c(II), M, 12,400, (charge 8+) reduction potential 260 mV, with parsley and French bean plastocyanins PCu(II) (charges — 7 and — 8 respectively), have been studied. As in the case of HIPIP, cytochrome c is not a physiologically relevant protein. It is nevertheless important in assessing different approaches prior to investigating the reactions of physiological redox partners. In the case of the reaction of parsley PCu(II) with cytochrome c(II), the rate constant (25 °C) is 1.5 X 10 s at pH7.6, 1 = 0.10 M(NaCl) [141]. There is no evidence... 

Parsley, spinach, French bean, poplar and S. obliquus (but not A. variabilis) conform extensively to the above criteria for reaction at the remote site. There is extensive evidence for cytochrome f reacting at the remote site on plastocyanin. The aromatic residue at 83 would seem to be a prime candidate as lead-in group for electron transfer. Desolvation at the surface around 83, and interaction with an aromatic component on the reaction partner, e.g. the porphyrin ring of cytochrome f, may be important. The exact manner of electron transfer has yet to be confirmed. The distance from the aromatic ring of Tyr83 to the Cu for electron transfer is 12 A. 

Early attempts at observing electron transfer in metalloproteins utilized redox-active metal complexes as external partners. The reactions were usually second-order and approaches based on the Marcus expression allowed, for example, conjectures as to the character and accessibility of the metal site. xhe agreement of the observed and calculated rate constants for cytochrome c reactions for example is particularly good, even ignoring work terms. The observations of deviation from second-order kinetics ( saturation kinetics) allowed the dissection of the observed rate constant into the components, namely adduct stability and first-order electron transfer rate constant (see however Sec. 1.6.4). Now it was a little easier to comment on the possible site of attack on the proteins, particularly when a number of modifications of the proteins became available. 

---