Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12 nm protein with interacting hemes

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Abstract

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2 Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15 Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c7-type domains (A, B, C and D) with homology to triheme cytochromes c7. In cytochromes c7 all three hemes are bis–His coordinated, whereas in c7-type domains the last heme is His–Met coordinated. The full-length GSU1996 has a 12 nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a “nanowire” of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c7-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c7-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c7-type domains from one multiheme protein were compared.

Introduction

Bacterial species important in biogeocycling of metals such as Fe encode large numbers of c-type cytochromes, with many proteins containing multiple heme c groups. One such bacterium is Geobacter sulfurreducens (Gs), well-suited to provide a deeper understanding of the role of individual multiheme cytochromes as its genome is fully sequenced (Methé et al., 2003), it is culturable in a laboratory, and a genetic system is available for making strains deficient in a protein of interest (Coppi et al., 2001). Gs can accept various electron donors and acceptors for survival suggesting it has versatile metabolic pathways. Such a premise would suggest a large number of redox proteins. Consistent with this scenario, the genome of Gs revealed that it encodes over 100 cytochromes c and several of them have been implicated in important roles in the respiration of this organism under various conditions (Shelobolina et al., 2007, Shi et al., 2007). For example, an outer membrane cytochrome, OmcB (a dodecaheme protein), is found to be essential for the reduction of insoluble Fe(III) oxide (Leang et al., 2003), a natural terminal electron acceptor for Gs in subsurface environments.

Apart from electron transfer roles, some multiheme cytochromes of Gs have also been suggested to be involved in gene regulation. Some evidence exists to indicate the regulatory roles of cytochromes OmcF, OmcG and OmcH in controlling the levels of OmcB expression and, thus, the levels of Fe(III) reduction (Kim et al., 2005, Kim et al., 2006). Another hypothesis proposed for the presence of large numbers of multiheme cytochromes in Gs is a possibility that these molecules may serve as electron-storage sinks or capacitors (Esteve-Núñez et al., 2008). As each heme can carry an electron, the multiheme proteins could hold many electrons collectively and allow the organism to survive in the absence of immediately available external electron acceptors. Such a capability would allow the continual generation of energy providing much needed extra time for the organism to relocate to new environments containing the external electron acceptors. Although several multiheme cytochromes have been identified to play critical roles in Fe(III) metabolism through knock-out mutant studies, the exact physiologic functions of most multiheme cytochromes of Gs are still unknown. The structure–function information for multiheme cytochromes is valuable in systems biology modeling of these bacteria that thrive in harsh conditions.

We had previously reported the structures of the five small triheme cytochromes c7 from Gs (Pokkuluri et al., 2004a, Pokkuluri et al., 2010). The cytochrome c7 is a small protein (70–75 amino acids) with three hemes covalently attached to the polypeptide through the CXXCH motif, and each heme has bis–His axial ligation. The hemes are named I, III and IV due to their structural homology to the equivalent hemes of tetraheme cytochromes c3 (Assfalg et al., 1998). Three larger multiheme proteins2 (two containing 12 hemes, GSU1996 and GSU0592, and one with 27 hemes, GSU2210) are also encoded in the Gs genome (Pokkuluri et al., 2004a). The sequences of these larger multiheme proteins can be divided into domains containing three hemes with homology to cytochromes c7. We refer to these domains as c7-type domains as they are different from cytochromes c7 mainly in the nature of the heme-IV axial ligation. In each of the c7-type domains, the hemes I and III have bis–His ligation but the heme-IV has His–Met axial ligation (Pokkuluri et al., 2004b). Proteins homologous to the above mentioned 12-heme and 27-heme cytochromes made up of multiple repeats of similar c7-type domains are also present in the genomes of other bacteria belonging to the Geobacteraceae family. The c7-type domains have high sequence homology (identities vary between 40% and 50%) within a given protein and also with domains from their homologs within Gs and from other Geobacteraceae. The sequence identities between the c7-type domains and cytochromes c7 is lower (∼30%). We had predicted that the c7-type domains in this novel family of multiheme cytochromes would be arranged linearly, connected by small linker regions like beads on a string (Pokkuluri et al., 2004a). A large multiheme cytochrome with modular structure composed of cytochrome c7 and cytochrome c3 domains has been reported. The structure of the high molecular weight cytochrome c (Hmc) from Desulfovibrio vulgaris Hildenborough containing 16-hemes revealed a structure composed of one c7 domain and three c3 domains (Czjzek et al., 2002, Matias et al., 2002).

To study the four domain multiheme cytochrome GSU19963, we had taken an approach to produce the proteins and determine the structures of the fragments in order of increasing sizes. First, we produced the individual domains containing three hemes, and determined the structure of one such c7-type domain (third domain from the N-terminus, domain C of GSU1996) at a resolution of 1.7 Å showing it to be a structural homolog of cytochrome c7 (Pokkuluri et al., 2004b).

In subsequent studies, we produced tandem two-domain halves of GSU1996 (hexaheme fragments termed AB, BC and CD; Londer et al., 2005) and also produced and crystallized the full-length molecules GSU1996 and GSU0592 (Londer et al., 2006). Recently, the reduction potentials of the individual hemes of domain C from GSU1996 were determined. Unexpectedly, they revealed that heme IV with His–Met coordination does not have the highest reduction potential (Morgado et al., 2009). In the present study, we report the structures of the hexaheme fragments AB and CD from GSU1996, and the structure of the full-length dodecaheme cytochrome, GSU1996. Having the structures of different fragments of a large multi-domain protein along with the structure of the full-length molecule allows us to evaluate the influence of the domain-domain interactions on the intrinsic structures of the individual domains.

Section snippets

Protein expression and purification

Expression and purification of the hexaheme fragments (Londer et al., 2005) and the full-length cytochromes GSU0592 and GSU1996 (Londer et al., 2006) were reported previously. Briefly, the proteins were produced in Escherichia coli strain JCB7123 (Gordon et al., 2001), except for domain AB that was produced in BL21(DE3). Both expression strains were co-transformed with cytochrome maturation plasmid pEC86 (Arslan et al., 1998).

For purification of BC and CD, the periplasmic fractions were

c7 type domain sequences

The sequence of GSU1996 can be divided into four tri-heme containing domains of 78–82 residues; they are designated as domains A, B, C, and D. The four domains have homologous amino acid sequences (Fig. 1). The amino acid sequence identities between the domains vary from 40% to 56% as shown in Table 2. There are 23 residues that are identical in all four domains (see Fig. 1), of which 12 are heme-binding residues (six Cys, five His and one Met). Of the remaining 11 residues, only one residue

Comparison of the c7-type domains with the c7 family

The c7-type domains from GSU1996 have sequences that are longer (78–82 residues) than the cytochromes c7 (70–75 residues). The N-terminal β-strands are a conserved feature of cytochromes c7 (Pokkuluri et al., 2010). This feature is observed in all of the four c7-type domains reported here. The loop connecting the N-terminal β-strands in each of the c7-type domains (except in domain D) is one residue longer than the corresponding segments in cytochromes c7. Another notable difference is that in

Summary

We have determined the structure of the multi-domain dodecaheme cytochrome GSU1996 at 3.2 Å resolution. Before a sample of full-length protein was available, we produced its fragments and determined their structures: single domain C at 1.7 Å resolution (Pokkuluri et al., 2004b), two-domain fragments, AB at 2.6 Å resolution and CD at 2.2 Å resolution. As is generally observed, the resolution of the diffraction data of the crystals decreased with increase in molecular size of the protein

Acknowledgments

The work at Argonne National Laboratory was supported by the US Department of Energy’s Office of Science, Biological and Environmental Research GTL program under contract No. DE-AC02-06CH11357 and by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-06CH11357. This work is a part of collaboration with Prof. D. R. Lovley (University of Massachusetts, Amherst) under the Genomics:GTL project. Use of the Structural Biology Center beam lines

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    Present address: New England Biolabs, 240 County Road, Ipswich, MA 01938, USA.

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