Crystal structure of Cytochrome cL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP^T

Abstract

Introduction Methylotrophic bacteria utilize methanol as their single carbon source of energy by oxidizing it to formaldehyde (Fig. 1) [1]. Methylotrophs are of great interest in the study of the biogeochemical cycling of methanol as well as the commercial production of complex polymers such as urea-formaldehyde resin and polyoxymethylene plastics [2]. The oxidation of methanol for energy in methylotrophic bacteria is carried out under the control of the methanol oxidizing (mox) operon, which consists of genes such as mxaF, mxaI, mxaJ, mxaG, mxaR, and mxaS, encoding α-and β-subunits of methanol dehydrogenase (MDH), MxaJ, cytochrome c L (Cytc L), MxaR, and MxaS, respectively (Fig. S1A) [3]. MDH comprises two subunits (α and β) and a cofactor pyrroloquinoline quinone (PQQ), which mobilizes two electrons from methanol, thereby converting it to formaldehyde [4]. The two electrons are subsequently transferred from PQQ of MDH to the heme of cytochrome c L through an unknown mechanism, followed by its sequential transfer to Cytc H , cytochrome c oxidase, and finally to ATP synthase in the cell membrane for the generation of ATP molecules (Fig. S1B) [5-8]. The electron transfer from MDH to Cytc L requires complex formation between these two proteins. Previous studies have proposed an interaction between the basic protein MDH and the acidic protein Cytc L for expedient electron transfer [6, 9, 10]. This complex formation is perplexing since it has never been isolated as a stable complex for crystallization, leading to the unavailability of a structure of the same till date. The docked protein complex [6] showed that the distance between the PQQ in the active site of MDH and the heme of Cytc L was approximately 20 Å, which was beyond the ideal electron jump distance (~14 Å) [11]. Furthermore, it was found that salt inhibits the initial complex formation and the subsequent electron transfer, and the inhibition is Cytochrome c L (Cytc L) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between Cytc L and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing Cytc L from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP T at 2.13 Å resolution. The crystal structure of Ma-Cytc L revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na + , Ca + , and Fe 2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive char...

Unusual Cytochrome c552 from Thioalkalivibrio paradoxus: Solution NMR Structure and Interaction with Thiocyanate Dehydrogenase

“…A group of topological homologues of CytC552 consists of four unusually large cytochromes c 551/552 (CytCL) from the periplasm of methylotrophic and denitrifying bacteria: Me -CytCL from Methylobacterium extorquens [ 22 ], Hd -CytCL from Hyphomicrobium denitrificans [ 23 ], Pd -CytCL from Paracoccus denitrificans [ 24 ], and Ma -CytCL from Methylophaga aminisulfidivorans [ 25 ] ( Figures S6 and S7 ). All CytCLs are electron acceptors for pyrroloquinoline quinone-containing methanol dehydrogenases (PQQ-MDH) [ 22 , 23 , 24 , 25 ]. Similar to TcDH, PQQ-MDH belongs to the class of β-propeller enzymes with catalytic sites located in the central tunnels of the β-propellers [ 26 , 27 ].…”

“…PQQ-MDH catalyzes oxidation of methanol to formaldehyde passing two electrons through periplasmic ET chain, which includes CytCLs and small soluble cytochromes c 2, to membrane-bound cytochrome oxidases [ 22 , 23 , 24 , 25 ]. Two models of putative PQQ-MDH/CytCL ET complexes were constructed using protein–protein docking [ 23 ] or homology modeling [ 24 ].…”