J Biol Inorg Chem B2002) 7: 23±30 DOI 10.1007/s007750100261 ORIGINAL ARTICLE Richard D. Kidd á Edward N. Baker á Thomas Brittain A modeling study of the interaction and electron transfer between cytochrome b 5 and some oxidized haemoglobins Received: 27 November 2000 / Accepted: 9 May 2001 / Published online: 27 June 2001 Ó SBIC 2001 Abstract Using Brownian motion simulations we have Introduction studied the formation of docked complexes of reduced cytochrome b and oxidized haemoglobin. Our results 5 In many biological processes, from the cytochrome indicate that the presence of molecular electrostatic ®elds chain of the mitochondrion to photosynthesis, a reaction has a signi®cant role to play in the formation of these of major importance is the deceptively simple process of complexes. In contrast to previous modeling studies on electron transfer. This process often involves the prior this system, we clearly identify electron transfer within formation of protein-protein complexes before the elec- an ensemble of similarly docked complexes rather than tron transfer event. Few such complexes have been iso- the formation of a single complex. Docking involves a lated and structurally characterized [1, 2] and questions number of acidic residues surrounding the exposed haem have been raised as to whether the few isolated com- edge of cytochrome b and a set of basic residues sur- 5 plexes are indeed of functional signi®cance [2, 3]. In the rounding the exposed haem edge of the globins. Al- absence of well-characterized electron transfer protein though amino acids from the partner globin proteins are complexes, theoretical approaches to the problem of involved to a small extent in the binding of some of the inter-protein electron transfer have been developed. In complexes, the reactivity of any particular globin is es- many cases, analyses of the formation of the initial sentially independent of the nature of its partner globin protein-protein complex have employed numerical sim- chain within the haemoglobin molecule. Comparison of ulation based on Brownian dynamic encounters modi- results from adult and embryonic haemoglobins indi- ®ed to take into account the eects of the electrostatic cates a signi®cant dierence in complex formation. Ap- ®elds associated with the protein partners [4, 5, 6]; this plication of electron tunneling analysis to the complexes approach appears to yield realistic models for the initial allows us to predict the rates of electron transfer within encounter for a number of systems [4, 5, 6, 7, 8]. How- each ensemble of complexes. These data provide a the- ever, more detailed investigations clearly require addi- oretical insight into the important process of re-reduc- tional analysis of such contributions as relaxation of the tion of oxidized haemoglobins as well as explaining the initial complex structures [9]. The analysis of the sub- general inability to produce crystalline forms of many sequent transfer of electrons from the donor protein to docked electron transfer complexes. the acceptor protein has often been based on the path- ways model developed by Onuchic and Beratan [10, 11], Keywords Cytochrome b5 á Haemoglobin á Brownian simulation á Electron transfer which analyses the process of electron tunneling between the partners in terms of the contributions of covalent bond, H-bond and through-space contributions, whilst explicitly taking into account the detailed structures of the redox partners. The original pathways model may in some circumstances also require modi®cation to take R.D. Kidd á E.N. Baker á T. Brittain B&) into account vibrational eects and the possible inter- School of Biological Science, ference between multiple dierent pathways [12, 13, 14, Private Bag 92019, 15, 16]. In other electron transfer systems, gating is also University of Auckland, recognized as a major factor modifying the overall Auckland, New Zealand electron transfer process between two proteins [17, 18, E-mail: [email protected] Tel.: +64-9-3737599 19, 20]. Thus in some systems the combination of Fax: +64-9-3737414 Brownian dynamics docking and pathways analysis 24 gives no more than a ``®rst-order approximation'' of the total process of inter-protein electron transfer. However, Materials and methods in the absence of any ®rm experimental data with which The crystallographic coordinates of cytochrome b , adult haemo- to make comparisons for the reaction of reduced cyto- 5 globin and embryonic haemoglobin Gower II were obtained from chrome b5 with met-haemoglobin, we have chosen, as a the Protein Data Bank B1CYO, 1BBB and 1A9W, respectively). ®rst step, to use this combination of approaches in our Disordered amino acids in the crystal structure of haemoglobin analysis of this system to make predictions concerning Gower II Ba22) were inserted using the graphics program O [36]. this important reaction. For haemoglobin Portland Bf2c2), the globin f chain was prepared by computer mutation of the a chain using O. The c chain struc- One particular electron transfer process is indirectly ture was obtained from the three-dimensional structure of hae- used to maintain the functional properties of haemo- moglobin Barts Bc4) BR.D. Kidd et al., in preparation). In all cases globin in its role as an oxygen carrying protein. In order where protein structures were modi®ed, the resulting protein to reversibly bind and release oxygen, the iron atom at structure was energy minimized using CNS [37]. All haemoglobin structures used were in the met Bferric) or isostructural ferrous the center of the haem groups of haemoglobin must liganded form. remain in its ferrous form. However, under physiologi- Simulations of electron transfer complex formation were per- cal conditions the oxy form of haemoglobin is intrinsi- formed using the software developed by Northrup and his col- cally unstable and can be oxidized to the non-functional leagues [4, 5, 6, 8, 38, 39]. The simulation process occurs in a ferric form by two distinct reactions. Mono-molecular number of related steps. The charge on the partner proteins Bre- duced cytochrome b5 and ferric haemoglobin) are ®rst calculated dissociation of the oxy-haemoglobin leads to the using Tanford-Kirkwood principles extended to include amino formation of superoxide and ferric haemoglobin [21]. acid solution accessibility [40, 41]. The resulting charge distribution Nucleophilic attack on the oxy form of haemoglobin also is then used to determine the electrostatic ®eld surrounding the leads to the formation of ferric haemoglobin [22]. The proteins with the Poisson-Boltzman equation using the Warwicker- Watson method [42]. Brownian motion of the partner proteins sum of these two reactions is responsible for approxi- within the electrostatic ®elds is then simulated by employing the mately 2% oxidation of adult human haemoglobin per Ermak and McCammon algorithm [43]. In order to obtain statis- day [23]. As the circulatory lifetime of a red blood cell is tically meaningful outcomes, 10,000 Brownian trajectories were approximately 120 days [24], it is necessary to constantly calculated for each simulation. Each simulation was performed on a dual processor SGI Octane and required approximately 9 h c.p.u. re-reduce the ferric haemoglobin to the ferrous form. time. All simulations were performed at an ionic strength of 0.1, a The reduction is achieved by a small electron transfer temperature of 25 °C and a pH of 6.2. Such simulations typically chain within the red blood cell, which leads from produced 400±700 electrostatically docked complexes. From the results of the docking simulations it is not only possible to identify NADH to cytochrome b5 reductase to cytochrome b5 and ®nally to ferric haemoglobin [25, 26]. The ®nal step the structures of the docked complexes but also the frequencies with which any particular amino acid participates in complex involves the transfer of an electron from cytochrome b5 formation. Furthermore, it is possible to predict a second-order to haemoglobin within a transient protein-protein rate constant for the electron transfer process. We have chosen to complex [27, 28]. By visual inspection of the structures use an exponential distance-dependent rate equation as a good of cytochrome b and isolated globin chains alone, approximation of the electron transfer process between two redox 5 centers contained within the partner proteins [44]. This model re- Poulos and Mauk [29] proposed a structure for a unique quires only the distance information generated from the Brownian cytochrome b5-a globin and cytochrome b5-b globin motion simulation and an appropriate distance decay factor [45]. complex. It was proposed that the binding within these In order to evaluate an appropriate distance decay factor for this complexes involved predominantly ionic interactions system, we randomly chose 50 individual complexes from the cy- tochrome b5-a2b2 trajectories and evaluated the eciency of elec- between surface Lys residues surrounding the haem tron transfer in each complex using the structure-speci®c analysis group on cytochrome b5 and Glu residues surrounding of electron tunneling in proteins developed by Onuchic and Ber- the haem site on the globin chains. Some support for atan [10, 46, 47, 48]. This set of data was then ®tted to an expo- this suggestion was gained from a small number of nential decay model to yield a value of the distance-dependent studies on the reactions of mutant and natural proteins decay factor, appropriate for this ensemble of complexes, that was then incorporated