Characterization of the haem environment in Methylophilus methylotrophus ferricytochrome c″ by ¹H‐NMR

Abstract

Two-dimensional NMR techniques have been used to assign proton resonances in the haem cavity of Methylophilus methylotrophus cytochrome c", a monohaem protein with bis-histidinyl ligation which has been shown to couple electron and proton transfer. All the assignments were made directly for the oxidized paramagnetic form of the cytochrome. Nearly all of the haem protons (90%) and the protons of both axial ligands have been assigned; the side-chain protons from four other residues in the haem pocket have also been identified. The data indicate a highly symmetric unpaired-electron distribution in the haem group, which agrees with a perpendicular orientation of the axial imidazole planes. The two haem propionate groups have contrasting degrees of exposure to the solvent, with the propionate group at position 13 being highly exposed. To obtain information on the dynamics of the haem environment, measurements of the 'W'H-exchange rates of amide protons located in the haem cavity were performed. The two faces of the haem are found to differ markedly with respect to water accessibility. All of this information, together with additional protein sequencing data, indicates that His52 remains attached upon reduction and that the redox-linked protonation occurs via a channel running through the haem cleft on the opposite face.Cytochrome c" is an unusual soluble monohaem cytochrome (15 kDa) isolated from the obligate methylotroph Methylophilus methylotrophus [l]. The sequence of 61 residues at the N-terminus of the protein is known, but no significant similarity has been found with the sequences of other proteins. However, the haem-attachment site shows the usual -CXXCH-sequence, typical of haem c proteins.NMR and ultraviolethisible spectroscopies have shown that the haem undergoes a redox-linked spin-state transition, going from a low-spin state in the oxidized form to a highspin state in the reduced form. The axial ligands are two histidine residues in the oxidized form, determined using magnetic CD analysis [2], and a single histidine residue in the reduced form determined using NMR analysis [l]. Furthermore, magnetic CD and EPR studies provided evidence for a near-perpendicular orientation of the two axial histidine residues in the oxidized form [2]. Ferricytochrome c" is a unique example of a haem-c protein with bis-histidine ligation and spectroscopic features similar to those found in model compounds in which the axial ligand planes are forced into a perpendicular orientation by steric constraints [3, 41. Correspondence to H . Santos, Instituto de Tecnologia QuimicaFax: +351 14428766. Abbreviations. NOESY, nuclear-Overhauser-effect spectroscopy; DQF-COSY, double-quantum-filtered proton-homonuclearshift correlation spectroscopy; TOCSY, total-correlation spectroscopy ; TI, longitudinal-relaxation time; COSY, correlation spectros-COPY.e Biolhgica, Apartado 127, P-2780 Oeiras, PortugalThe midpoint redox potential of cytochrome c" shows a strong pH dependence (redox-Bohr effect) in the physiological pH range, and some of t...

Crystal Structures of an Oxygen-binding Cytochrome cfrom Rhodobacter sphaeroides

“…Compared with the proximal side, the distal heme plane is more solvent accessible, a conclusion similar to that based on NMR data for the homologous M. methylotrophus cytochrome cЉ (15). The amino acid providing the sixth heme ligand was found to be Asn…”

“…Recently, cytochrome cЉ from the obligate methylotroph Methylophilus methylotrophus was shown to be 32% identical to SHP (15,16), and a 42% identical open reading frame is present in the genome of the metal oxidereducing bacterium Shewanella putrefaciens (Fig. 1).…”

pH Dependence of Structural and Functional Properties of Oxidized Cytochrome c" from Methylophilus methylotrophus

“…Cytochrome cЉ is a unique example of a heme-c protein with bis-histidine coordination and spectroscopic features similar to those observed in model compounds where axial ligand planes are forced into a perpendicular orientation by steric constraints (3). NMR studies of the heme pocket have shown that it is quite stable at neutral pH, with low amide proton exchange rates and one of the heme propionates largely exposed at the surface, whereas the other one is buried in the protein (1). Attempts to crystallize the protein have been unsuccessful to date.…”

“…Cytochrome cЉ from the obligate methylotroph Methylophilus methylotrophus is a soluble monoheme protein of ϳ15 kDa, which displays a redox-linked spin state transition from a low spin state in the oxidized form to a high spin state in the reduced form (1). The two axial ligands are histidines in the oxidized form, one of which is detached from heme upon reduction of the iron atom (2).…”

“…The pH dependence of the midpoint redox potential has been analyzed in terms of a model that considers two ionizing groups with pK a values that change with the redox state of the protein (1,4). It was concluded that the group mainly responsible for the redox Bohr effect exhibited by cytochrome cЉ is one of the axial histidines that becomes detached in the reduced state with a pK a Х 8.1 for the ⑀ 2 -NH in the reduced form and a pK a Ͻ 2 in the oxidized form (1,4). However, this very low pK a could not be determined accurately, because its value was far outside the range investigated by NMR and redox potentiometry.…”