Neisseria gonorrhoeae (Ng) is a human pathogen of growing concern owing to the impact of Ng infections on human health [1,2] and increasing spread of antibiotic-resistant Ng strains [2,3]. The ability of Ng to efficiently transition between denitrification and aerobic respiration enables this pathogen to thrive in a variety of environments [4]. The sole cytochrome (cyt) c oxidase in Ng is a cbb3-type oxidase that catalyzes oxygen reduction and proton pumping at its active site under aerobic conditions [5,6], therefore this enzyme and the proteins it interacts with may serve as powerful antibiotic targets. The diheme protein cytochrome c4 (c4) has been suggested as an electron donor to cbb3 oxidase in multiple bacteria, acting alone or together with another heme protein, cytochrome c5 (c5), to transfer electrons from the bc1 complex to cbb3 oxidase [[7], [8], [9], [10]]. In Ng, mutations in genes for c4 and c5 have revealed increased growth inhibition of the bacteria when placed in highly aerated environments [9]. A strain having deletions of both c4 and c5 was not possible to isolate under aerobic conditions, and this result has been interpreted to suggest that these two proteins are involved in electron transfer to cbb3 oxidase [9].

Genomic analysis showed widespread presence of c4 proteins within proteobacteria [8]. Solution properties of multiple c4 proteins have been reported [8,[11], [12], [13], [14], [15], [16], [17]], but at this point, structural information is only available for c4 proteins from Pseudomonas stutzeri (Ps) [18], Pseudomonas aeruginosa (Pa) [19], and Acidithiobacillus ferrooxidans (Af) [20]. Ps c4 is the most well-characterized and several of its variants have been examined [21].

In Ps c4, the two hemes differ in their reduction potentials by ∼100 mV, which is thought to be governed by the distinct surface charges of the two heme domains [18]. A similar relationship between potentials and surface charges exists for c4 proteins from several other species [8,12,19]. For some c4 proteins in which the two heme groups are equipotential, such as ones from Pa [19] and Pseudoalteromonas haloplanktis (Ph) [15], no distinct charge patches are present. At the same time, the two hemes in both Pa and Ph c4 differ in their solvent accessible surface area (SASA) [19]. The interdomain interface has been suggested to also influence potentials of c4 proteins [19,20], but its effects are complex. Recent efforts in our group have uncovered that the interdomain interface in Ps c4 plays a role in folding of the c4-B (C-terminal) domain, prevents homodimerization of the c4-A (N-terminal) domain, and increases potentials of both folded c4-A and c4-B by 120 mV by stabilizing the ferrous forms of the domains [21].

Herein, we report the solution properties and crystal structure of Ng c4. The two hemes of Ng c4 greatly differ in their solvent exposure. The structure reveals a prominent positively-charged patch, which encompasses surfaces of both c4-A and c4-B domains. We correlate these structural features with reduction potentials of the heme centers, determine the contribution of the interdomain interface to potentials of the isolated domains, and suggest implications for reactions of c4 with its putative redox partners.